SUMO-dependent transcriptional repression by Sox2 inhibits the proliferation of neural stem cells

Elisa Marelli; Jaime Hughes; Paul J Scotting

doi:10.1371/journal.pone.0298818

. 2024 Mar 20;19(3):e0298818. doi: 10.1371/journal.pone.0298818

SUMO-dependent transcriptional repression by Sox2 inhibits the proliferation of neural stem cells

Elisa Marelli ^1,^*, Jaime Hughes ¹, Paul J Scotting ¹

Editor: Anujith Kumar²

PMCID: PMC10954124 PMID: 38507426

Abstract

Sox2 is known for its roles in maintaining the stem cell state of embryonic stem cells and neural stem cells. In particular, it has been shown to slow the proliferation of these cell types. It is also known for its effects as an activating transcription factor. Despite this, analysis of published studies shows that it represses as many genes as it activates. Here, we identify a new set of target genes that Sox2 represses in neural stem cells. These genes are associated with centrosomes, centromeres and other aspects of cell cycle control. In addition, we show that SUMOylation of Sox2 is necessary for the repression of these genes and for its repressive effects on cell proliferation. Together, these data suggest that SUMO-dependent repression of this group of target genes is responsible for the role of Sox2 in regulating the proliferation of neural stem cells.

Introduction

All members of the Sox protein family share a high level of sequence similarity and function as transcription factors [1]. They are characterized by a conserved HMG DNA-binding domain composed of 79 amino acids that contains the conserved sequence motif “RPMNAFMVW”. Sox proteins are able to recognize and bind to the consensus sequence “5’-WWCAAW-3’” (where W = A/T) [2]. This binding process causes a widening of the minor groove that leads to the bending of the DNA structure [3–5]. It has been proposed that Sox factors function as architectural transcription factors that recruit other protein through their binding to the DNA; the proteins recruited would be other transcription factors, chromatin re-modelers or other regulatory partners that would act as co-activators (or co-repressors) of target genes.

SoxB proteins, which include Sox1, Sox2, Sox3, Sox14 and Sox21, play a central role in several processes during the embryonic development of vertebrates and insects [4, 6–8]. Sox1, Sox2 and Sox3 belong to the SoxB1 subgroup and exhibit a high level of full-length sequence similarity and some functional redundancy. SoxB1 transcription factors are thought to play a major role in the regulation of the pluripotent state of neural stem cells (NSC) in the central nervous system (CNS) [9]. The expression of SoxB1 genes firstly marks ectodermal cells, which have the potential to become neural cells and later expression becomes restricted to ectodermal cells committed to a neural fate. This has been shown in zebrafish, Drosophila, Amphioxus, Xenopus and chicken embryos [10–18].

SoxB1 factors have been shown to play roles in both determining neural fate and in maintaining the multipotency of NSCs [13, 19]. In chicken embryos, the differentiation of neural progenitors was prevented by over-expression of Sox2 and/or Sox3. Importantly, these cells exhibited features of more undifferentiated cells such as the ability to proliferate and the expression of early neural progenitor markers [20, 21]. On the other hand, over-expression of dominant-negative forms of Sox2 and/or Sox3 caused premature exit from the cell cycle and the induction of neuronal differentiation. A study by Kondo et al. (2004) showed that through re-initiation of Sox2 expression it is possible to convert mouse oligodendrocyte progenitor cells (OPCs) back into multipotent neural stem-like cells [22]. The same study showed that inhibition of Sox2 caused premature exit from the cell cycle and differentiation of the OPCs towards neural fate [9, 23]. In mouse, Sox2 is expressed in undifferentiated stem and progenitor cells in the CNS and its expression decreases with the progression of differentiation, although it remains expressed in some populations of differentiated neurons [24]. Cavallaro et al. (2008) created Sox2 knockdown mutants in mice and studied in vitro differentiation of embryonic and adult NSCs. They showed that neuronal differentiation was affected in mutant cells and that this condition could be rescued by over-expression of Sox2 in the early stages of differentiation. According to these data, the activity of Sox2 is therefore crucial for the differentiation of NSC towards neuronal identity. However, it seems that mice are much less affected by defects in the expression of Sox2 compared to humans [25, 26] highlighting how much Sox2 activity and function are context dependent.

Genome-wide single cell analysis, followed by functional analysis in mouse cortex showed that when stem cells transition into rapidly dividing progenitor cells, the expression of Sox2 is reduced [27]. According to this study, stem cells express high levels of Sox2, which represses proliferative genes such as Cyclin D1 through binding of low-affinity DNA motifs. Conversely, as these cells differentiate Sox2 expression is reduced and so expression of Cyclin D1 is derepressed, promoting proliferation.

SoxB1 factors have been generally considered to exert their functions by activating the expression of target genes. However, more recent evidence suggests that SoxB1 factors can be either transcriptional activators or repressors and that their function is highly dependent on the context of when and where they are expressed [28, 29]. This context-dependency could be correlated with post-transcriptional modifications and changes in the interacting partners of the SoxB1 factors.

As for most members of the Sox family, the activity of Sox2, whether it is as an activator or a repressor, is dependent upon the co-factor recruited. In fact, Sox2 is expressed in different developmental and cellular contexts, where it exhibits different activities. Therefore, it is important to define which are the target genes of Sox2 in each context and how Sox2 regulates these targets in different contexts. According to a model proposed by Remenyi et al. (2003), Sox2 can partner with a specific co-factor assuming different conformational arrangements depending on the distance between the two binding sites on the DNA [30]. Therefore, Sox2 may elicit tissue-specific functions by switching its interaction partners. However, it is still not clear how these switches are regulated and whether post-translational modifications could play a role in these mechanisms by altering which cofactors Sox2 interacts with.

We postulate that the switch between the activating and repressing activity of Sox2 could be regulated by post-translational modification. In fact, Sox2 is subject to a number of post-translational modifications such as phosphorylation [31–34], methylation [31, 35], acetylation [36], ubiquitination [31] and SUMOylation [34, 37]. Little is currently known about Sox2 SUMOylation and its function. SUMOylation has been shown to regulate transcription factors’ activity through a variety of mechanisms [38].

Sox2 is SUMOylated at lysine 247 in vivo and SUMOylation is lost when the target lysine is substituted with an arginine (K247R) [37]. This study showed that endogenous Sox2 was SUMOylated in human gastric cancer cells and that the protein’s ability to activate the Fgf4 minimal enhancer in cooperation with the co-activator Oct3/4 was inhibited by SUMOylation. However, it is yet to be determined whether such an effect of Sox2 SUMOylation is general or specific to the Fgf4 enhancer [39]. In fact, SUMOylation of a transcription factor can have the opposite effect, for example it has been shown that SUMOylation of Oct4 increases the protein’s stability, DNA binding and transactivation [40, 41]. Wu et al. (2012) showed that SUMOylated Sox2 inhibited the expression of the transcription factor Nanog, while SUMOylated Oct4 increased the expression of Nanog. They proposed a model according to which SUMOylation interferes with the formation of the Oct4-Sox2 heterodimer, which is required to regulate Nanog.

Because SUMOylation is known to be involved in the regulation of the activity of other transcription factors, and since Sox2 has been shown to act both as a transcriptional activator and repressor, we set out to test the hypothesis that SUMOylation of Sox2 could regulate the function of Sox2 by altering the balance between its transcriptional activation and transcriptional repression activity. This hypothesis is supported by studies of other Sox factors. For example, SoxE protein could induce neural crest development, while a SoxE SUMO-fusion repressed neural crest formation in Xenopus embryos [42]. Lee et al. (2012) also showed that SUMOylated SoxE protein loses the ability to bind the co-activator CBP/p300 and recruit the co-repressor Grg4.SUMOylation has also been shown to affect the activity of the SoxB1 transcription factor SoxNeuro in Drosophila, which is structurally and functionally homologous to Sox1, Sox2 and Sox3 [43, 44]. SUMOylation of the central nervous system specific SoxB proteins has been conserved during evolution as Sox2 and Sox3 are also targeted by SUMOylation. The mutation of human Sox3 SUMO-targeted lysine to arginine increased Sox3 transcriptional activation activity, suggesting that SUMOylation could inhibit Sox3 transcriptional activation activity [45].

By comparing the effects of over-expressing exogenous wt Sox2, non-SUMOylatable Sox2 mutant or constitutively SUMOylated Sox2 in human neural stem cells (hNSC), we found that SUMOylated Sox2, similarly to wt (SUMOylatable) Sox2, caused the downregulation of a set of proliferation related genes. Many of these genes have been published as Sox2 targets in Chromatin Immunoprecipitation Sequencing (ChIP-Seq) assays. Moreover, we show that SUMOylated Sox2 or wt (SUMOylatable) Sox2 also slow the proliferation of hNSCs. Both of these effects were lost when the SUMO acceptor site of Sox2 was mutated.

Materials and methods

Design and cloning of Sox2 mutant constructs

The following Sox2 mutant constructs were constructed. constructedSox2_N48I contains an Asparagine to Isoleucine mutation in position 48, which is in the HMG DNA-binding domain. Sox3 has been shown to be unable to bind the DNA helix when containing an N40I mutation [46–48]. Given the sequence similarity and functional redundancy of Sox2 and Sox3, we hypothesized that the equivalent point mutation in Sox2 would have had the same effect. A sequence alignment revealed that the Asparagine 40 residue present in Sox3 is conserved in Sox2 and corresponds to Asparagine 48. We therefore expected Sox2_N48I not to be able to bind to the DNA. Sox2_NLS is expected not to be translocated into the nucleus due to six amino acids changes (K44T, R45G, R58L, R59G, R115L and R116G) in both of the two presumptive Nuclear Localization Signal sites of the protein [47, 49]. Sox2_HMG-eng contains the Sox2 HMG domain fused to the engrailed repressor domain. This construct is expected to act as a constitutive transcriptional repressor [50, 51]. Sox2_HMG-VP16 is a fusion protein containing the HMG domain of Sox2 fused to the VP16 activator domain [50]. This construct is expected to behave as a transcriptional activator. Sox2_Δgrg contains mutations in the binding site of Groucho (grg), which is a co-repressor of Sox2, and is expected to prevent Groucho binding [29] by altering four amino acids in positions 203, 207, 208 and 209 (D, L, Q and Y to V, A, A and A). This mutant Sox2 protein has been shown to inhibit the differentiation of NSC to a lesser extent compared to wild-type Sox2 (wt Sox2) [29]. Sox2-SUMO1, Sox2-SUMO2 and Sox2-SUMO3 are fusion proteins containing the wt Sox2 entire protein fused at its C-terminus with SUMO1, SUMO2 or SUMO3 peptides. It has been reported that SUMO conjugation at the target lysine or at the carboxyl terminus of a target protein have a similar effect [37, 52]. The use of C-terminally fused SUMO peptides has been found to mimic the constitutively SUMOylated state of a protein and it has been shown to be a useful approach especially when the SUMOylation site is near the C-terminus of the protein, as it is for Sox2 [42, 52–54]. However, these fusion proteins could potentially be double SUMOylated once transfected into cells as the proteins SUMOylation site (lysine 247) is intact. Sox2_K247A is mutated in the presumptive SUMOylation site [37] and is expected not to be SUMOylated once transfected into cells. In fact, lysine 247 (K247) has been reported to be targeted by SUMOylation [34, 37, 55, 56]. Sox2_K247R is also mutated in the same position of Sox2_K247A and is expected to have lost the ability to be SUMOylated endogenously [37, 56]. Literature reports that mutating lysine residues targeted by SUMO into arginine very often results in a complete or near complete abrogation of the SUMOylation of the target protein [57]. Sox2_K247R-SUMO1, Sox2_K247R-SUMO2 and Sox2_K247R-SUMO3 are fusion proteins containing Sox2_K247R fused at his C-terminus with SUMO1, SUMO2 or SUMO3 peptides. As the SUMO-acceptor lysine is mutated to an arginine, these proteins should not be SUMOylated by the cellular SUMOylation machinery. Therefore, this should prevent the presence of two SUMO peptides, one fused to the C-terminus and the other attached to the SUMO acceptor site, on the protein.

Wt Sox2, Sox2_K247R, Sox2_K247A, Sox2_N48I, Sox2_NLS, Sox2_Δgrg, were obtained through site-directed mutagenesis using QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies 210518) and QuikChange Lightning Multi-Site Directed Mutagenesis Kit (Agilent 210515) following manufacturer’s instructions.

Western blot

Running gel (12.5% w/v of polyacrylamide) was prepared according to the following recipe, poured between two gel plates in a gel castor and covered with 70% EtOH: 2.14 ml Acryl-Bis 29:1, 1.43 ml 1.57 M Tris HCl pH 8.8 and 0.4% SDS, 1.44 ml water, 75 μl APS 10%, 7.5 μl TEMED. After removal of EtOH a stacking gel (12.5% w/v of polyacrylamide) was prepared according to the following recipe and poured over polymerized running gel: 0.65 ml Acryl-Bis 29:1, 1.25 ml 0.5 M Tris HCl pH 6.8 and 0.4% SDS, 1.73 ml water, 75 μl APS 10%, 7.5 μl TEMED. After solidification the gels were transferred into the gel tank, which was then filled with SDS Running Buffer (30.3g Tris, 188g Glycine, 10ml 10% SDS (in sterile deionized water), sterile deionized water up to 1L).

Sample buffer (Laemmli 2x concentrate, Sigma-Aldrich S3401-10VL) was diluted 1:1 with PBS and added to the frozen cell pellets before running SDS-PAGE. Samples were then boiled at for 10 minutes and incubated on ice for 10 minutes before loading on SDS gel. The protein ladder used for all the experiments of the present study was the SeeBlue Plus2 Pre-Stained Protein Standard (Life Technologies LC5925) and the amount loaded was always 5μl.

The gels were run at 40V until all the samples have reached the running gel, and then at 80V until completion of SDS-PAGE running. The gels were then blotted on Amersham Protran Premium western blotting membrane (GE Healthcare GE10600118) using the following transfer buffer: 200ml MeOH, 3.03 ml Tris, 14.4 ml Glycine, sterile deionized water up to 1L.

The transfer was run at 30 V overnight. The membrane was then removed and immersed into 5% Marvel Milk in PBS with 0.01% Tween20 (PBST) for at least 1 hour at room temperature, with gentle shaking. The primary antibodies were diluted at the appropriate concentration in PBS added with 0.01% Tween20 (Sigma-Aldrich) (PBST) and used to soak the membrane overnight at 4 ˚C gently shaking. Following the probing, the membrane was washed in PBST for 5 minutes 3 times while gently shaking, it was then probed with secondary antibody diluted 1/5000 in PBST and incubated for at room temperature for at least 1 hour gently shaking and then washed in PBST for 5 minutes 3 times.

Primary antibodies used: Mouse anti-Sox2 (20G5) Thermo Scientific MA1-014 used (diluted 1:2000), Rabbit anti-HA tag Abcam ab236632 (diluted 1:5000), Mouse anti-Myc tag (9E10) Abcam ab32 (diluted 1:4000). Secondary antibodies used: Donkey anti-mouse IRDye 800CW Li-Cor 926–32212 (diluted 1:5000), Goat anti-rabbit IRDye 680LT Li-Cor 926–68021 (diluted 1:10,000)

Subcellular fractionation

Buffer A: 10 mM HEPES (pH 7.9), 10 mM KCl, 1.5 mM MgCl2, 0.34 M sucrose, 1 mM dithiothreitol, 10% Glycerol and distilled water. Buffer B: ethylenediaminetetraacetic acid (EDTA) 3mM, ethylene glycol tetra acetic acid (EGTA) 0.2 mM, dithiothreitol 1 mM and distilled water. Protease inhibitor cocktail (Sigma-Aldrich) was added to both buffer A and buffer B just before use. Frozen cell pellets were washed twice with ice cold PBS and centrifuged each time at 1800 xg at 4°C for 2 minutes. Cell pellets were then re-suspended in 100 μl of buffer A with 0.1% of TritonX-100 added (Sigma-Aldrich 11332481001) and incubated on ice for 8 minutes. The samples were then centrifuged at 1300 x g at 4°C for 5 minutes: the supernatants were collected, and the pellets were discarded. The fractions collected were clarified by high-speed centrifugation at 20,000 xg at 4°C for 5 minutes. The supernatants (cytosolic fractions) were collected and placed on ice, while the pellets were washed once with buffer A and lysed by addition of 50 μl of buffer B followed by incubation on ice for 30 minutes. The samples were then centrifuged at 1700 xg at 4°C for 5 minutes and the supernatants (nuclear fractions) were collected and placed on ice while the pellets (chromatin fractions) were washed once with 50 μl of buffer B. All the subcellular fractions were stored at -80°C.

Culture and transfection of hNSC

ReNcell VM neural progenitor cells (Merck Millipore SCC008) were used in this study as an in vitro model of hNSC. They were grown either as neurospheres or as adherent monolayer on laminin coated plasticware in the following culture medium: 50% DMEM:F12 (GIBCO 11554546), 50% Neurobasal (GIBCO 21103049), 0.5% Penicillin-Streptomycin (Sigma-Aldrich P4333-100ML), 1% N2 supplement (GIBCO 17502048), 2% B27 NeuroMix (GIBCO 17504044), heparin (5μg/ml, Sigma-Aldrich 9041-08-1). Human Epidermal Growth Factor (hEGF, GIBCO AF-100-15-1MG) and human Fibroblast Growth Factor (hFGF, GIBCO 100-18B-1MG) were added to the cell culture medium just before use, both at a concentration of 20 ng/ml. Growth medium was replaced with fresh medium every 2 days.

Transfection of hNSC was performed by electroporation using a Nucleofector^TM 2b Device (Lonza) with Nucleofector^TM Kits for Mouse Neural Stem Cells as per manufacturer instructions (Lonza VPG-1004).

Luciferase reporter assay

HEK-293 cells were grown in Dulbecco’s Modified Eagle Medium (DMEM, Sigma-Aldrich) with 10% foetal bovine serum (FBS, Sigma-Aldrich F2442) and penicillin-streptomycin (Sigma-Aldrich P4333-100ML). Cells were seeded in black 96-well plates and cultured for 24 hours. On the following day, cells were transfected with 25 ng of reporter and 150 ng of activator using Turbofect (Thermo Scientific R0531) and incubated in 5% CO₂ for 24 hours. On the following day, culture medium was removed from each well and replaced with 80 μl of 1x Lysis buffer (Dual-Luciferase Reporter Assay System, Promega E1910). Plates were incubated at room temperature for 10 minutes gently rocking and then each sample was pipetted 10 times before transferring the plates to the GloMax luminometer (Promega) for the reading. The luminometer was set to inject 25 μl of Luciferase Reagent for each well with a delay of 2 seconds between injection and measuring.

RNA sequencing and data analysis

Human NSC were cultured as monolayers in T-75 flasks until 70% confluent, co-transfected with pcDNA3, wt Sox2, Sox2_K247R or Sox2_K247R-SUMO2 and GFP and seeded in fresh T-75 flasks. The day after transfection, the cells were rinsed with PBS and fresh growth medium added. The following day, cells were harvested and suspended in 660 ml of fresh growth medium. For each sample 160 ml of cell suspension was removed and used for western blot. The remaining 500 ml were run through Beckman Coulter MoFlo XDP to isolate only GFP-expressing cells by Fluorescence-Activated Cell Sorting (FACS). Non-transfected hNSC were used as negative control for cell sorting.

RNA was purified using the RNeasy Plus Mini Kit (QIAGEN 74134) as per manufacturer’s instructions. Purified RNA was eluted with 50 ml RNase-free water.

RNA quantity control was performed by Nanodrop (Thermo Scientific TM1000). Quality control was performed using the Agilent 2100 Bioanalyzer System.

The experiment was repeated 3 times, generating 12 samples which were then analysed though RNA sequencing.

RNA samples were sent for RNA sequencing to the Oxford Genomics Centre based at the Wellcome Centre for Human Genetics (Department of Medicine, University of Oxford). They prepared 12 libraries and ran them on 2 lanes of the HiSeq4000 using 75bp PE reads. The libraries were prepared using Illumina’s TruSeq Stranded mRNA Library Prep Kit as per manufacturer’s specifications with custom primers. Custom primers were used for the PCR enrichment step: Multiplex PCR primer 1.0:

5’-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’Index primer:

5’-CAAGCAGAAGACGGCATACGAGAT[INDEX]CAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3’Indices were designed according to the eight base tags developed by WTCHG (70).

Homo_sapiens.GRCh38.dna_sm.primary_assembly.fa reference genome and Homo_sapiens.GRCh38.84.gff3 annotation file were downloaded from:

ftp://ftp.ensembl.org/pub/release-84/fasta/homo_sapiens/dna.

Differential Expression analysis was performed in R using DESeq2.

Cell proliferation assay

Transfected cells were seeded into two 96-well plates and cultured for 7 days. Medium was replaced every 2 days. Each day 3 replicate wells per transfected sample were analysed using an Alamar Blue cell proliferation assay (Invitrogen DAL1025). One set of wells was used to take pictures daily and to obtain images showing the cell confluency day by day. The entire experiment was repeated 3 times (biological replicates). Wells were analysed 24 hours after the addition of Alamar Blue.

Cell death assay

Cell death was detected and measured using CellEvent™ Caspase-3/7 Green Detection Reagent assay (Invitrogen C10423). Human NSC were cultured in a T75 flask for a few passages, harvested and counted before transfection. Cells used for the different samples came from the same population. Cells were then treated with transfection reagent only, cultured with G418 (Sigma G8168-10ML), or transfected with pcDNA3, wt Sox2, Sox2_K247R or Sox2_K247R-SUMO2 DNA. Cells from each sample were then used both for the visual assessment experiment and for the first replica of the quantitative experiment (measurement of apoptotic events using a plate reader). After transfection, hNSC were plated both in 6-well plates containing glass coverslips coated with poly-D-lysine and laminin (4x10⁵ cells/well) for the visual testing experiment and in black 96-well plates with optical clear bottom for the quantitative assessment. All the samples were incubated O/N at 37°C and the analyses started on the following day (day 1). NSCs cultured O/N with increasing doses of G418 presented increasing amounts of fluorescent cells, confirming that the apoptotic assay can detect apoptotic cells (S1 Fig).

In order to determine how long the samples should be incubated, every set of samples was analysed 30 min, 1 h, 2 h, 24 h and then every 24h until day 5 after addition of CellEvent. The results showed that incubation for 24h showed the highest differences between positive and negative controls (S2 Fig).

Results

Sox2 SUMOylation inhibits its transcriptional activation ability

In order to investigate whether SUMOylation affects the transcriptional activity of Sox2, we first tested the ability of Sox2 to activate the expression of a target gene in vitro using a Firefly luciferase activation reporter assay. In these experiments, the expression of the Firefly luciferase gene was regulated by an artificial upstream Sox promoter containing three Sox2 binding sites [29]. The activities of wild-type Sox2 (wt Sox2) and several different Sox2 mutant constructs (S3 Fig) were compared to test whether targeted mutations altered the protein’s transcriptional activation function (Fig 1). The activity of the Sox2 constructs was compared to non-transfected cells (UN) and cells transfected with reporter gene only (luciferase). wt Sox2 successfully activated the luciferase reporter gene. As negative controls, we generated and tested a Sox2 mutant construct with a point mutation designed to prevent DNA binding (N48I) as well as a construct mutated at the nuclear localisation site (NLS). As expected, both these constructs were unable to activate the reporter gene. We also generated constitutive repressor (eng) and constitutive activator (VP16) forms of Sox2 by fusing the Sox2 HMG DNA-binding domain to either an engrailed repressor domain or VP16 activator domain. These constructs behaved as expected, with the eng construct lacking activation function and the VP16 construct activating the reporter gene to a similar extent to wt Sox2. Sox2_ΔGrg (a Sox2 construct mutated at the Groucho binding site and therefore incapable of binding to the co-repressor Groucho (35)) did not show any significant difference compared to the positive control. Six different SUMO fusion constructs, which carry SUMO1/2/3 peptides fused to the C-terminus of either wt or the K247R sumo mutant forms of Sox2 and are therefore constitutively SUMOylated (Sox2_SUMO1/2/3 and Sox2-K247R_SUMO1/2/3), gave a significantly lower signal compared to Sox2-VP16, while two ‘SUMO mutants’ (Sox2-K247A and Sox2-K247R in which the SUMO-acceptor Lysine was mutated) resulted in a significantly higher activation of the reporter gene (Fig 2).

Fig 1 — Average readings of three independent biological replicates together shows that wt Sox2 is statistically different (t test) from the SUMOylation mutant Sox2 constructs (D). Constitutive SUMOylation had an opposite effect compared to the absence of SUMOylation. Data are normalized to the positive control HMG_VP16 (constitutive activator). Statistical differences were calculated using unpaired t test. Bars indicate the standard error of the mean.

Fig 2 — Wt Sox2 can be SUMOylated at K247. The Sox2_K247R construct carries a point mutation that prevents SUMOylation. Conversely, Sox2_K247R-SUMO2 is constitutively SUMOylated at the N-terminus.

These results suggest that the transcriptional activation ability of Sox2 is reduced in vitro when the protein is SUMOylated. This is consistent with results in the literature [37], where a Sox2-SUMO1 fusion construct was shown to lose its ability to activate the fgf4 minimal enhancer together with the co-factor Oct3/4.

The luciferase reporter assay showed that SUMOylation appears to inhibit the activation activity of Sox2. However, this assay only assessed the ability of Sox2 to activate expression when driven by one particular artificial promoter in HeLa cells and did not assess transcriptional repression. Therefore, the effects of SUMOylation on Sox2 transcriptional activity were further analysed using a genome-wide approach in human hNSCs.

SUMOylation of Sox2 is necessary for the protein’s transcriptional repression activity

Since fusion of Sox2 to all three SUMO subtypes had a similar effect in the luciferase assay described above, we used one of these to study the effect of SUMOylation in more detail. The transcriptional activity of wt Sox2, Sox2_K247R and Sox2_K247R-SUMO2 was investigated by transfection of hNSCs followed by RNA sequencing. Over-expression of the transfected constructs was assessed by western blot (S4 Fig) The expression levels of the exogenous constructs achieved with our transfection protocol was comparable to the levels of endogenous Sox2 (S5 Fig). The experiment was repeated three times (three biological replicates) for each condition (transfection of empty vector, wt Sox2, Sox2_K247R, Sox2_K247R-SUMO2) generating 12 samples, which were analysed by RNA sequencing.

Analysis of genes differentially expressed in comparison to control transfected cells (vector only) showed a reduction in the transcriptional activity of Sox2_K247R, the over-expression of which affected only 3.5% of all genes differentially expressed (34 out of 971), compared to wt Sox2 (affected 43% of the genes, 419 out of 971) or the SUMO fusion construct Sox2_K247R-SUMO2 affecting 74% of all genes differentially expressed (722 out of 971) (Fig 3). Overall, the number of down-regulated genes identified (69%) was higher compared to up-regulated genes (31%). Over-expression of wt Sox2 in hNSCs repressed more genes (320) than it activated (99). Similarly, over-expression of constitutively SUMOylated Sox2 (Sox2_K247R-SUMO2) in hNSCs repressed more genes (503) than it activated (219). On the contrary, over-expression of non-SUMOylatable Sox2 (Sox2_K247R) in hNSCs activated more genes (21) than it repressed (13).

Fig 3 — Total number of genes differentially expressed (A). Number of genes whose expression was significantly decreased (B) and number of genes whose expression was significantly increased (C).

A high proportion of the genes affected by these Sox2 constructs (190 genes, approximately 20% of all differentially expressed genes identified) were common between hNSC over-expressing wt Sox2 and those over-expressing the sumo fusion construct, Sox2_K247R-SUMO2 (3). Most of these genes (25% of all the downregulated genes identified, 165 out of 667) were repressed and none of these shared repressed genes were also repressed in hNSC over-expressing the non-SUMOylatable Sox2_K247R construct (3). This suggests a functional similarity between exogenous wt Sox2, which can be endogenously SUMOylated at the K247 site, and constitutively SUMOylated construct Sox2_K247R-SUMO2 as opposed to non SUMOylatable Sox2_K247R. In fact, approximately 52% of the genes repressed by wt Sox2 are also repressed by Sox2_K247R-SUMO2 (165 out of 320). Conversely, only 0.6% of the genes repressed by wt Sox2 are also repressed by Sox2_K247R SUMO-deficient construct, suggesting that Sox2 loses its transcriptional repression activity in absence of SUMOylation. Since the Sox2_K247R mutant construct loses the ability to affect the genes that are repressed by wt Sox2, this implies that the effect seen upon expression of exogenous wt Sox2 on these genes is due to that proportion of exogenous Sox2 that becomes SUMOylated. Hence, the reason why the rescued mutant construct (Sox2_K247R-SUMO2) has a stronger transcriptional regulation effect than the wt Sox2, is likely to be because all of the protein produced is now SUMOylated. Together, these data imply that the effects of the expression of these constructs on the genes identified is predominantly due to the SUMOylation of Sox2. This is consistent with the published literature, which reports that, despite a small proportion of SUMO target proteins being actually SUMOylated, it is often the SUMOylated portion of the protein that regulates most of the transcriptional activity of the protein. This phenomenon has been termed the ‘SUMO enigma’ [58, 59].

The data obtained on repressed genes is in contrast with the data on activated genes, most of which are unique to each construct, with only approximately 22% of the genes activated by wt Sox2 also activated by Sox2_K247R-SUMO2 (22 genes out of 99). This suggests that SUMOylation directly affects Sox2 transcriptional activity causing the downregulation of several target genes.

Since our experiments relied on the function of exogenous Sox2 constructs, we compared the genes affected to those identified as targets of endogenous Sox2 via Chip-Seq analysis. Comparison to the published studies in mouse ESCs and NSCs revealed that 97 of the 165 genes downregulated in our studies were identified as direct Sox2 targets using Chip-Seq analysis. Together with our observation that the levels of exogenous Sox2 appears to be comparable to the level of endogenous Sox2 (S5 Fig), this suggests that, although it’s possible that the endogenous Sox2 could affect some non-physiological targets, SUMOylation of endogenous Sox2 is also likely to play a role in its ability to repress target genes.

SUMO-dependent Sox2 repressed target genes are involved in the regulation of the cell cycle

Gene ontology term enrichment analysis for ‘cellular process’ (GOrilla, http://cbl- gorilla.cs.technion.ac.il/) performed on the 165 genes down-regulated when either wt Sox2 oror Sox2_K247R-SUMO2 were over-expressed (but not repressed in hNSC over-expressing_K247R) showed an enrichment in genes ‘regulating cell cycle process’ and ‘mitotic cell cycle process’ (Table 1). Moreover, gene ontology analysis performed on the same set of genes based on their ‘molecular function’ revealed an enrichment for ‘nucleic acid binding’ and ‘microtubule binding’ functions (Table 2). The same type of analysis performed on the 25 genes up-regulated in hNSC over-expressing wt Sox2 and in hNSC over-expressing Sox2_K247R did not show enrichment of genes involved in any particular cellular process or molecular function.

Table 1. Results of gene ontology analysis of genes down-regulated in hNSC over-expressing wt Sox2 or Sox2_K247R-SUMO2 based on cellular process.

P value	Cellular process
< 10⁻⁹	• Mitotic cell cycle process
10⁻⁷ to 10⁻⁹	• Regulation of mitotic cell cycle • Cell cycle process • Regulation of mitotic cell cycle phase transition • mRNA splicing • mRNA processing

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Table 2. Results of gene ontology analysis of genes down-regulated in hNSC over-expressing wt Sox2 or Sox2_K247R-SUMO2 based on molecular function.

P value	Molecular function
< 10⁻⁹	• Nucleic acid binding • RNA binding
10⁻⁷ to 10⁻⁹	• Tubulin binding • Microtubule binding

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Protein interaction analysis on the same 165 genes using String software (https://string-db.org/) showed two clusters of genes involved in mitotic cells cycle processes (Fig 4). One of these two clusters (Fig 4, bottom right) included the genes CEP 152 (codifying for Centrosomal Protein 152), CEP250 (codifying for Centrosome-Associated Protein CEP250), CEP290 (codifying for Centrosomal Protein 290), CENPJ (codifying for Centromere Protein J), CNTRL (codifying for 152Centriolin), CDK5RAP2 (codifying for CDK5 Regulatory Subunit Associated Protein 2), AKAP9 (codifying for A-Kinase Anchoring Protein 9) and GOLGA2 (codifying for Golgin A2). All of these genes are involved in mitotic processes including microtubule organisation, centrosome assembly and centromere integrity.

The other gene cluster identified (Fig 4, bottom left) included CENPE (Centrosome-Associated Protein E), CENPF (codifying for Centromere protein F), CLIP1 (codifying for CAP-Gly Domain Containing Linker Protein 1), INCEMP (codifying for Inner Centromere Protein), SMC1A (Structural Maintenance of Chromosomes 1A), SPAG5 (codifying for Sperm Associated Antigen 5), KIF15 (Kinesin Family Member 5) and KIF18b (Kinesin Family Member 18b). These genes are also involved in the formation of the mitotic spindle and in the regulation of mitosis. This suggests that SUMOylation of Sox2 is required for the transcriptional repression of many target genes involved in the regulation of mitosis. In the same comparison with published Chip-Seq data used above, 22 of the 32 cell cycle-related genes downregulated in our studies were identified as direct Sox2 targets. Therefore, the data presented suggest that SUMOylation of Sox2 plays a role in the regulation of hNCS proliferation.

SUMOylation of Sox2 affects hNSC proliferation

Results obtained in this study suggest that SUMOylation of Sox2 may be important in its ability to regulate hNSC proliferation. Moreover, in all experiments that involved transient over-expression of either wt Sox2 or Sox2-SUMO fusion constructs for two or more days the cell density was noticeably lower than in other samples. Based on this observation, we hypothesised that SUMOylation of Sox2 might be necessary for Sox2 to repress NSC proliferation and that the absence of SUMOylation might ablate this effect. In order to investigate this hypothesis, we set out to compare the proliferation rates of hNSCs over-expressing either pcDNA3 (vector only), wt Sox2, Sox2_K247R or Sox2_K247R-SUMO2 (Fig 5). Proliferation rates were measured either 4 hours (S6 Fig) or 24 hours after transfection (Fig 5).

Fig 5 — Proliferation rate, measured by Alamar Blue proliferation assay. Bars indicate standard error of the mean. Three biological replicates were performed. Within each biological replicate, three technical replicates were performed.

Cells transfected with either pcDNA3, wt Sox2, Sox2_K247R or Sox2_K247R-SUMO2 showed a growth curve that reached its plateau around day 6 after transfection before reaching the plateau, samples transfected with either wt Sox2 or Sox2_K247R-SUMO2 grew at a slower rate compared to the control (pcDNA3). The rate of growth of cells transfected with Sox2_K247R was almost the same as the control.

Statistical comparison between the proliferation rates of the 4 samples showed that the proliferation rate of cells over-expressing either wt Sox2 or Sox2_K247R-SUMO2 was significantly lower than the negative control (pcDNA3) at days 2, 3 and 4 (Fig 6A). These differences tended to disappear 6 days after transfection, presumably due to degradation of the transfected plasmid DNA and its encoded protein and to the cells reaching confluency. At day 7 after transfection, there was a slight decrease in the proliferation detected, presumably due to cell death and cell detachment caused by high cell confluency (Fig 6B, panel d7).

Fig 6 — (A). Statistical differences were calculated using unpaired t test. Bars indicate standard error of the mean. Three biological replicates (Cell Proliferation 1, 2 and 3) were performed. Within each biological replicate, three technical replicates were performed. Pictures of hNSC over-expressing the different Sox2 constructs and cultured for 7 days (B).

Images taken in parallel with the proliferation assays clearly illustrate how cells transfected with wt Sox2 or Sox2_K247R-SUMO2 appeared as confluent as cells transfected with pcDNA3 on day 1 (24 hours after transfection and seeding), but then appeared less confluent for the following days, until approximately day 5 and 6, when they reached a high confluency comparable to that of the control (Fig 6B).

SUMOylation does not affect apoptosis in hNSC

The data above suggest that SUMOylation of Sox2 could contribute to the regulation of NSC proliferation rate. Such a hypothesis is consistent with the results obtained through RNA sequencing. However, the apparent effect on proliferation could be due to apoptosis induced by over-expression of wt Sox2 or Sox2_K247R-SUMO2. In order to investigate this, we set out to compare the rate of apoptosis in cells transfected with these constructs to samples transfected with pcDNA3 or Sox2_K247R.

Cell death was analysed both on cells grown on microscope coverslips and then visualised after treatment with CellEvent (visual assessment) and on cells grown in a 96-well plate, treated with CellEvent and analysed using a plate reader (quantitative assay).

Samples transfected with either pcDNA3, wt Sox2, Sox2_K247R or Sox2_K247R-SUMO2 presented very few slightly fluorescent cells, barely visible in the photographic images, with no clear difference in numbers between these samples. By contrast, most positive control cells treated with 10 g/ml G418 were clearly fluorescent (S1 Fig)

For quantitative analysis of cell death, cells were transfected on day 0 and fluorescence was measured every 24 hours. No significant difference between samples was detected during the course of the experiment, until the faster proliferating samples reached complete confluency, when faster growing cells exhibited increase fluorescence earlier than slower growing samples.

The results of these experiments suggest that transfection of hNSCs with either wt Sox2 or Sox2_K247R-SUMO2 affected the cells’ proliferation rate rather than affecting their viability (Fig 7).

Fig 7 — Apoptosis was measured over the course of 6 days. Two biological replicates of the experiment were performed. Within each biological replicate, two technical replicates were performed. Bars indicate standard deviation of the mean.

SUMOylation does not affect Sox2 subcellular localisation

We next set out to determine whether the effect that SUMOylation of Sox2 has on the protein’s transcriptional activation activity could be due to a change in its subcellular localization. Sub-cellular fractionation of hNSCs over-expressing wt Sox2, Sox2-SUMO2, Sox2_K247R or Sox2_K247R-SUMO2 was followed by western blot of the isolated cellular fractions.

Quantitative analysis by infrared fluorescence imaging confirmed that the wt Sox2 protein was mainly localized in the chromatin and in the cytosol in all cases. Both Sox2_SUMO2 and Sox2_K247R-SUMO2 fusion proteins in the chromatin and cytosol fractions at 3 to 6 fold higher levels than Sox2 and Sox2_K234R, while still presenting a similar overall sub-cellular distribution (Fig 8). This indicates that the SUMO fusion constructs are either more highly expressed or more stable than the other Sox2 constructs analyzed. Sox2-SUMO2 produced a brighter band compared to wt Sox2 30h after transfection of hNSC (S4 Fig), which could suggest that the Sox2 SUMO2-fusion protein is more stable over time. Thus, although the overall level of protein varied for different constructs, subcellular localization of Sox2 was not affected by SUMOylation.

Fig 8 — (A). The average amounts of proteins in different sub-cellular compartments are shown relative to the amounts of wt Sox2 or relative to the amounts of Sox2_K247R present in each sub-cellular compartment (B). Bars indicate standard error of the mean.

Discussion

It is well-established that Sox2 plays a central role in the biology of neural stem cells by maintaining their stemness and regulating their proliferation and differentiation [9, 27, 60]. Our study suggests that SUMOylation of Sox2 is central to its ability to regulate neural stem cell proliferation and that this is largely due to a requirement for SUMO-dependent repression of target genes associated with mitotic cell cycle processes.

Our data suggest that SUMOylation of Sox2 by SUMO2 does not alter Sox2 sub-cellular localisation, consistent with studies reported in the literature [34, 56]. We also found that the level of over-expressed SUMOylated Sox2 in hNSCs was higher than over-expressed wt Sox2. This is consistent with previous western blots performed in our laboratory on hNSC at different times after transfection and suggests that Sox2_SUMO2 might be more stable than wt Sox2 and Sox2_K247R. The hypothesis that SUMOylation could stabilise Sox2 is consistent with reports that SUMO can antagonise ubiquitin and therefore prevent a protein’s degradation [61–63]. The results obtained through RNA sequencing are also consistent with this hypothesis as over-expression of Sox2_K247R-SUMO2 affected the expression of more genes (722) than did wt Sox2 (419). This could be explained by the relative stability of each of these constructs: if the Sox2 SUMO2-fusion protein was more stable than the other Sox2 constructs, then its effects on target genes would also be more stable over time, resulting in a higher number of genes being affected at the time when expression levels were analysed, 48 hours after transfection.

Since only a small proportion of Sox2 (probably less than 5%) is normally SUMOylated, the fact that a non-SUMOylatable mutant loses almost all transcriptional activity implies that it is this small fraction of SUMOylated wt Sox2 that is transcriptionally active. If the effect of this mutant was non-specific (not due to loss of SUMOylation) then it would not be rescued by the fusion of SUMO to the mutant’s C-terminus. However, it is clear that much of this activity is indeed rescued by addition of a fused SUMO peptide (despite the fact that the construct still carries the same K to R mutation) and this is particularly evident for repressed genes.

Over-expression of wt Sox2 or Sox2_K247R-SUMO2 altered the expression of the same 190 genes, representing approximately 20% of the total number of genes differentially regulated by either of the two constructs. The vast majority of these common genes were repressed (87%). Conversely, out of the genes differentially expressed in cells over-expressing wt Sox2 or Sox2_K247R, only 4% were common and 82% of these were transcriptionally activated by Sox2_K247R. This suggests that the repression of most Sox2 target genes is SUMO-dependent and can be ablated by the lack of the SUMOylation site, K247. This evidence supports the initial hypothesis that SUMOylation of Sox2 affects the protein by enhancing its transcriptional repression activity.

Our results suggest that SUMOylation of Sox2 is essential for its ability to regulate the proliferation rate of hNSCs as, similar to hNSCs over-expressing wt Sox2, cells over-expressing SUMOylated Sox2 grew at a slower rate than controls and down-regulated the same set of mitotic progression regulating genes. Conversely, hNSCs over-expressing Sox2_K247R proliferated at a rate similar to negative control cells and exhibited almost no transcriptional repression activity. This is consistent with Hagey and Muhr (2014) who showed that in mouse embryonic cortical tissue Sox2 represses genes that promote neural precursor cell proliferation. The slow proliferation of NSCs is of upmost importance for their biological role and for preserving the stem cells pool of an organism over time [64]. Therefore, the hypothesis that SUMOylation of Sox2 may be directly involved in the regulation of such a process could have a major impact on the current knowledge of hNSC biology.

Because the activity of Sox2 is tightly linked to the recruitment of co-factors, a possible model that would explain the effect of SUMOylation of Sox2 is that SUMOylated Sox2 could recruit specific co-repressors to repress target cell cycle-related genes. Therefore, if Sox2 loses the ability to be SUMOylated, it also loses the ability to interact with these co-repressors. This model could be tested by protein precipitation purifying Sox2 and its interaction partners, which could then be identified through mass spectrometry.

The importance of the role that Sox2 plays in regulating the pluripotency and the proliferation of NSCs is well known even though the underlying mechanisms of action are still being discovered. It is also known that Sox2 is a versatile transcription factor with the ability of transcriptionally activate and repress different sets of target genes in different cellular and developmental contexts. The mechanisms regulating such functional versatility, while maintaining high target specificity, are also still unclear.

The present study obtained evidence that SUMOylation of Sox2 plays a central role in promoting the function of Sox2 as a transcriptional repressor. This is consistent with the literature, as deficiency of the SUMOylation enzyme, Ubc9, has recently been found to enhance reprogramming of mouse embryonic fibroblasts into induced pluripotent stem cells both in vivo and in vitro and that SUMOylation functions as a general barrier to cell-fate transitions [65].

We also noted that 34 of the genes differentially regulated by both wt Sox2 and constitutively SUMOylated Sox2_K247R-SUMO2 are involved in human syndromes linked to microcephaly. Of these, 33 are down-regulated by both constructs while one was up-regulated (S1 Table). Since mutations in the closely related SoxB1 gene, Sox3, are associated with human syndromes that include microcephaly, the repression of these target genes might therefore also provide a link between loss of Sox3 activity and microcephaly.

Sox2 has been extensively studied and yet there is much more to uncover on how it is functionally regulated within different cellular contexts. The data shown in the present study open a completely new insight into the molecular mechanisms that regulate the transcriptional activity of one of the major players in stem cell fate determination. These data suggest previously unknown mechanisms that can now be pursued in further studies as it is clear that SUMOylation has a major effect on the regulation of particular Sox2 target genes and consequently on stem cell proliferation.

Supporting information

S1 Fig. Visual assessment of apoptosis assay performed on hNSC grown in the presence of G418 (positive controls) or transfected with different Sox2 constructs and cultured O/N.

Scale bars: 50 μm.

(TIFF)

pone.0298818.s001.tiff^{(3.5MB, tiff)}

S2 Fig. Measurements of apoptosis on day 1 sets of wells incubated for different amounts of time after addition of CellEvent.

Two biological replicates were performed. Within each biological replicates, two technical replicates were performed. Bars indicate standard deviation of the mean.

(TIFF)

pone.0298818.s002.tiff^{(385KB, tiff)}

S3 Fig. Expression of Sox2 constructs in HeLa cells.

Membrane was probed using anti-Myc primary antibody followed by green Licor secondary antibodies.

(TIFF)

pone.0298818.s003.tiff^{(1.4MB, tiff)}

S4 Fig. Western blot performed on hNSC to assess the expression of Sox2 constructs used for RNA Sequencing.

Anti-Myc antibody was used to detect exogenous Sox2 proteins.

(TIFF)

pone.0298818.s004.tiff^{(452.2KB, tiff)}

S5 Fig. Western blot performed on lysates of hNSC transfected with wt Sox2 or Sox_K247R or co-transfected with wt Sox2 or Sox2_K247R, SUMO2 and Ubc9.

All the Sox2 constructs transfected are Myc-tagged and they are the same constructs used thought the present study. The membrane was firstly probed with mouse anti-Myc and rabbit anti-HA primary antibodies and green LiCor anti-mouse and red LiCor anti-rabbit secondary antibodies (A). The same membrane was then probed again using mouse anti-Sox2 primary antibodies and green Licor anti-mouse secondary antibodies (B). The size difference between exogenous and endogenous Sox2 is due to the exogenous Sox2 constructs containing Myc and His tags.

(TIFF)

pone.0298818.s005.tiff^{(1.8MB, tiff)}

S6 Fig. Proliferation of hNSC after transient transfection with either pcDNA3, wt Sox2, Sox2_K247R or Sox2_K247R-SUMO2 and incubation with Alamar Blue for 4 hours.

Bars indicate standard error of the mean.

(TIFF)

pone.0298818.s006.tiff^{(335.5KB, tiff)}

S1 Table. Genes differentially expressed by wt Sox2 and Sox2_K247R-SUMO2 associated with human microcephaly.

(TIF)

pone.0298818.s007.tif^{(451.7KB, tif)}

S1 File. Minimal data sets underlying the results presented and original western blot images.

(XLSX)

pone.0298818.s008.xlsx^{(2.4MB, xlsx)}

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

EM - The present work was funded through BBSRC-Doctoral Training Partnership Programme (PhD) awarded to Elisa Marelli. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

1.Laudet V, Stehelin D, Clevers H. Ancestry and diversity of the HMG box superfamily. Nucleic Acids Res. 1993;21(10):2493–501. Epub 1993/05/25. doi: 10.1093/nar/21.10.2493 ; PubMed Central PMCID: PMC309552. [DOI] [PMC free article] [PubMed] [Google Scholar]
2.Harley VR, Lovell-Badge R, Goodfellow PN. Definition of a consensus DNA binding site for SRY. Nucleic Acids Res. 1994;22(8):1500–1. Epub 1994/04/25. doi: 10.1093/nar/22.8.1500 ; PubMed Central PMCID: PMC308012. [DOI] [PMC free article] [PubMed] [Google Scholar]
3.Ferrari S, Harley VR, Pontiggia A, Goodfellow PN, Lovell-Badge R, Bianchi ME. SRY, like HMG1, recognizes sharp angles in DNA. EMBO J. 1992;11(12):4497–506. Epub 1992/12/01. doi: 10.1002/j.1460-2075.1992.tb05551.x ; PubMed Central PMCID: PMC557025. [DOI] [PMC free article] [PubMed] [Google Scholar]
4.Lefebvre V, Dumitriu B, Penzo-Mendez A, Han Y, Pallavi B. Control of cell fate and differentiation by Sry-related high-mobility-group box (Sox) transcription factors. Int J Biochem Cell Biol. 2007;39(12):2195–214. Epub 2007/07/13. doi: 10.1016/j.biocel.2007.05.019 ; PubMed Central PMCID: PMC2080623. [DOI] [PMC free article] [PubMed] [Google Scholar]
5.Kamachi Y, Cheah KS, Kondoh H. Mechanism of regulatory target selection by the SOX high-mobility-group domain proteins as revealed by comparison of SOX1/2/3 and SOX9. Mol Cell Biol. 1999;19(1):107–20. Epub 1998/12/22. doi: 10.1128/MCB.19.1.107 ; PubMed Central PMCID: PMC83870. [DOI] [PMC free article] [PubMed] [Google Scholar]
6.Phochanukul N, Russell S. No backbone but lots of Sox: Invertebrate Sox genes. Int J Biochem Cell Biol. 2010;42(3):453–64. Epub 2009/07/11. doi: 10.1016/j.biocel.2009.06.013 . [DOI] [PubMed] [Google Scholar]
7.Soriano NS, Russell S. The Drosophila SOX-domain protein Dichaete is required for the development of the central nervous system midline. Development. 1998;125(20):3989–96. Epub 1998/09/15. doi: 10.1242/dev.125.20.3989 . [DOI] [PubMed] [Google Scholar]
8.Uchikawa M, Kamachi Y, Kondoh H. Two distinct subgroups of Group B Sox genes for transcriptional activators and repressors: their expression during embryonic organogenesis of the chicken. Mech Dev. 1999;84(1–2):103–20. Epub 1999/09/03. doi: 10.1016/s0925-4773(99)00083-0 . [DOI] [PubMed] [Google Scholar]
9.Pevny L, Placzek M. SOX genes and neural progenitor identity. Current opinion in neurobiology. 2005;15(1):7–13. Epub 2005/02/22. doi: 10.1016/j.conb.2005.01.016 . [DOI] [PubMed] [Google Scholar]
10.Cremazy F, Berta P, Girard F. Sox neuro, a new Drosophila Sox gene expressed in the developing central nervous system. Mechanisms of development. 2000;93(1–2):215–9. doi: 10.1016/s0925-4773(00)00268-9 . [DOI] [PubMed] [Google Scholar]
11.Hardcastle Z, Papalopulu N. Distinct effects of XBF-1 in regulating the cell cycle inhibitor p27(XIC1) and imparting a neural fate. Development. 2000;127(6):1303–14. doi: 10.1242/dev.127.6.1303 . [DOI] [PubMed] [Google Scholar]
12.Holland LZ, Schubert M, Holland ND, Neuman T. Evolutionary conservation of the presumptive neural plate markers AmphiSox1/2/3 and AmphiNeurogenin in the invertebrate chordate amphioxus. Developmental biology. 2000;226(1):18–33. doi: 10.1006/dbio.2000.9810 . [DOI] [PubMed] [Google Scholar]
13.Mizuseki Kishi, Matsui Nakanishi, Sasai. Xenopus Zic-related-1 and Sox-2, two factors induced by chordin, have distinct activities in the initiation of neural induction. Development. 1998;125(4):579–87. doi: 10.1242/dev.125.4.579 [DOI] [PubMed] [Google Scholar]
14.Penzel R, Oschwald R, Chen Y, Tacke L, Grunz H. Characterization and early embryonic expression of a neural specific transcription factor xSOX3 in Xenopus laevis. Int J Dev Biol. 1997;41(5):667–77. . [PubMed] [Google Scholar]
15.Rex M, Orme A, Uwanogho D, Tointon K, Wigmore PM, Sharpe PT, et al. Dynamic expression of chicken Sox2 and Sox3 genes in ectoderm induced to form neural tissue. Dev Dyn. 1997;209(3):323–32. Epub 1997/07/01. doi: . [DOI] [PubMed] [Google Scholar]
16.Russell SR, Sanchez-Soriano N, Wright CR, Ashburner M. The Dichaete gene of Drosophila melanogaster encodes a SOX-domain protein required for embryonic segmentation. Development. 1996;122(11):3669–76. doi: 10.1242/dev.122.11.3669 . [DOI] [PubMed] [Google Scholar]
17.Streit A, Sockanathan S, Perez L, Rex M, Scotting PJ, Sharpe PT, et al. Preventing the loss of competence for neural induction: HGF/SF, L5 and Sox-2. Development. 1997;124(6):1191–202. doi: 10.1242/dev.124.6.1191 . [DOI] [PubMed] [Google Scholar]
18.Uwanogho D, Rex M, Cartwright EJ, Pearl G, Healy C, Scotting PJ, et al. Embryonic expression of the chicken Sox2, Sox3 and Sox11 genes suggests an interactive role in neuronal development. Mech Dev. 1995;49(1–2):23–36. Epub 1995/01/01. doi: 10.1016/0925-4773(94)00299-3 . [DOI] [PubMed] [Google Scholar]
19.Pevny LH, Sockanathan S, Placzek M, Lovell-Badge R. A role for SOX1 in neural determination. Development. 1998;125(10):1967–78. Epub 1998/06/18. doi: 10.1242/dev.125.10.1967 . [DOI] [PubMed] [Google Scholar]
20.Bylund M, Andersson E, Novitch BG, Muhr J. Vertebrate neurogenesis is counteracted by Sox1-3 activity. Nat Neurosci. 2003;6(11):1162–8. Epub 2003/10/01. doi: 10.1038/nn1131 . [DOI] [PubMed] [Google Scholar]
21.Graham V, Khudyakov J, Ellis P, Pevny L. SOX2 functions to maintain neural progenitor identity. Neuron. 2003;39(5):749–65. Epub 2003/09/02. doi: 10.1016/s0896-6273(03)00497-5 . [DOI] [PubMed] [Google Scholar]
22.Kondo T, Raff M. Chromatin remodeling and histone modification in the conversion of oligodendrocyte precursors to neural stem cells. Genes Dev. 2004;18(23):2963–72. doi: 10.1101/gad.309404 ; PubMed Central PMCID: PMC534656. [DOI] [PMC free article] [PubMed] [Google Scholar]
23.Wegner M, Stolt CC. From stem cells to neurons and glia: a Soxist’s view of neural development. Trends Neurosci. 2005;28(11):583–8. doi: 10.1016/j.tins.2005.08.008 . [DOI] [PubMed] [Google Scholar]
24.Cavallaro M, Mariani J, Lancini C, Latorre E, Caccia R, Gullo F, et al. Impaired generation of mature neurons by neural stem cells from hypomorphic Sox2 mutants. Development. 2008;135(3):541–57. doi: 10.1242/dev.010801 . [DOI] [PubMed] [Google Scholar]
25.Fantes J, Ragge NK, Lynch SA, McGill NI, Collin JR, Howard-Peebles PN, et al. Mutations in SOX2 cause anophthalmia. Nat Genet. 2003;33(4):461–3. Epub 2003/03/04. doi: 10.1038/ng1120 . [DOI] [PubMed] [Google Scholar]
26.Sisodiya SM, Ragge NK, Cavalleri GL, Hever A, Lorenz B, Schneider A, et al. Role of SOX2 mutations in human hippocampal malformations and epilepsy. Epilepsia. 2006;47(3):534–42. doi: 10.1111/j.1528-1167.2006.00464.x . [DOI] [PubMed] [Google Scholar]
27.Hagey DW, Muhr J. Sox2 acts in a dose-dependent fashion to regulate proliferation of cortical progenitors. Cell reports. 2014;9(5):1908–20. doi: 10.1016/j.celrep.2014.11.013 . [DOI] [PubMed] [Google Scholar]
28.Chew LJ, Gallo V. The Yin and Yang of Sox proteins: Activation and repression in development and disease. J Neurosci Res. 2009;87(15):3277–87. doi: 10.1002/jnr.22128 ; PubMed Central PMCID: PMC2767240. [DOI] [PMC free article] [PubMed] [Google Scholar]
29.Liu YR, Laghari ZA, Novoa CA, Hughes J, Webster JR, Goodwin PE, et al. Sox2 acts as a transcriptional repressor in neural stem cells. BMC Neurosci. 2014;15:95. doi: 10.1186/1471-2202-15-95 ; PubMed Central PMCID: PMC4148960. [DOI] [PMC free article] [PubMed] [Google Scholar]
30.Remenyi A, Lins K, Nissen LJ, Reinbold R, Scholer HR, Wilmanns M. Crystal structure of a POU/HMG/DNA ternary complex suggests differential assembly of Oct4 and Sox2 on two enhancers. Genes Dev. 2003;17(16):2048–59. doi: 10.1101/gad.269303 ; PubMed Central PMCID: PMC196258. [DOI] [PMC free article] [PubMed] [Google Scholar]
31.Fang L, Zhang L, Wei W, Jin X, Wang P, Tong Y, et al. A methylation-phosphorylation switch determines Sox2 stability and function in ESC maintenance or differentiation. Mol Cell. 2014;55(4):537–51. doi: 10.1016/j.molcel.2014.06.018 . [DOI] [PubMed] [Google Scholar]
32.Jeong CH, Cho YY, Kim MO, Kim SH, Cho EJ, Lee SY, et al. Phosphorylation of Sox2 cooperates in reprogramming to pluripotent stem cells. Stem Cells. 2010;28(12):2141–50. doi: 10.1002/stem.540 . [DOI] [PubMed] [Google Scholar]
33.Swaney DL, Wenger CD, Thomson JA, Coon JJ. Human embryonic stem cell phosphoproteome revealed by electron transfer dissociation tandem mass spectrometry. Proc Natl Acad Sci U S A. 2009;106(4):995–1000. doi: 10.1073/pnas.0811964106 ; PubMed Central PMCID: PMC2633571. [DOI] [PMC free article] [PubMed] [Google Scholar]
34.Van Hoof D, Munoz J, Braam SR, Pinkse MW, Linding R, Heck AJ, et al. Phosphorylation dynamics during early differentiation of human embryonic stem cells. Cell Stem Cell. 2009;5(2):214–26. doi: 10.1016/j.stem.2009.05.021 . [DOI] [PubMed] [Google Scholar]
35.Zhao HY, Zhang YJ, Dai H, Zhang Y, Shen YF. CARM1 mediates modulation of Sox2. PLoS One. 2011;6(10):e27026. Epub 2011/10/28. doi: 10.1371/journal.pone.0027026 ; PubMed Central PMCID: PMC3203945. [DOI] [PMC free article] [PubMed] [Google Scholar]
36.Baltus GA, Kowalski MP, Zhai H, Tutter AV, Quinn D, Wall D, et al. Acetylation of sox2 induces its nuclear export in embryonic stem cells. Stem Cells. 2009;27(9):2175–84. doi: 10.1002/stem.168 . [DOI] [PubMed] [Google Scholar]
37.Tsuruzoe S, Ishihara K, Uchimura Y, Watanabe S, Sekita Y, Aoto T, et al. Inhibition of DNA binding of Sox2 by the SUMO conjugation. Biochem Biophys Res Commun. 2006;351(4):920–6. Epub 2006/11/14. doi: 10.1016/j.bbrc.2006.10.130 . [DOI] [PubMed] [Google Scholar]
38.Rosonina E, Akhter A, Dou Y, Babu J, Sri Theivakadadcham VS. Regulation of transcription factors by sumoylation. Transcription. 2017;8(4):220–31. Epub 2017/04/06. doi: 10.1080/21541264.2017.1311829 ; PubMed Central PMCID: PMC5574528. [DOI] [PMC free article] [PubMed] [Google Scholar]
39.Kondoh H, Lovell-Badge R. Sox2 Biology and Role in Development and Disease: Academic Press; 2016. [Google Scholar]
40.Wei F, Scholer HR, Atchison ML. Sumoylation of Oct4 enhances its stability, DNA binding, and transactivation. The Journal of biological chemistry. 2007;282(29):21551–60. Epub 2007/05/26. doi: 10.1074/jbc.M611041200 . [DOI] [PubMed] [Google Scholar]
41.Zhang Z, Liao B, Xu M, Jin Y. Post-translational modification of POU domain transcription factor Oct-4 by SUMO-1. FASEB J. 2007;21(12):3042–51. Epub 2007/05/15. doi: 10.1096/fj.06-6914com . [DOI] [PubMed] [Google Scholar]
42.Taylor KM, Labonne C. SoxE factors function equivalently during neural crest and inner ear development and their activity is regulated by SUMOylation. Dev Cell. 2005;9(5):593–603. Epub 2005/11/01. doi: 10.1016/j.devcel.2005.09.016 . [DOI] [PubMed] [Google Scholar]
43.Wegner M. From head to toes: the multiple facets of Sox proteins. Nucleic Acids Res. 1999;27(6):1409–20. Epub 1999/02/26. doi: 10.1093/nar/27.6.1409 ; PubMed Central PMCID: PMC148332. [DOI] [PMC free article] [PubMed] [Google Scholar]
44.Bowles J, Schepers G, Koopman P. Phylogeny of the SOX family of developmental transcription factors based on sequence and structural indicators. Dev Biol. 2000;227(2):239–55. Epub 2000/11/10. doi: 10.1006/dbio.2000.9883 . [DOI] [PubMed] [Google Scholar]
45.Savare J, Bonneaud N, Girard F. SUMO represses transcriptional activity of the Drosophila SoxNeuro and human Sox3 central nervous system-specific transcription factors. Mol Biol Cell. 2005;16(6):2660–9. Epub 2005/03/25. doi: 10.1091/mbc.e04-12-1062 ; PubMed Central PMCID: PMC1142414. [DOI] [PMC free article] [PubMed] [Google Scholar]
46.Kuo CL, Lam CM, Hewitt JE, Scotting PJ. Formation of the embryonic organizer is restricted by the competitive influences of Fgf signaling and the SoxB1 transcription factors. PLoS One. 2013;8(2):e57698. Epub 2013/02/28. doi: 10.1371/journal.pone.0057698 ; PubMed Central PMCID: PMC3585176. [DOI] [PMC free article] [PubMed] [Google Scholar]
47.Shih YH, Kuo CL, Hirst CS, Dee CT, Liu YR, Laghari ZA, et al. SoxB1 transcription factors restrict organizer gene expression by repressing multiple events downstream of Wnt signalling. Development. 2010;137(16):2671–81. doi: 10.1242/dev.054130 . [DOI] [PubMed] [Google Scholar]
48.Zhang C, Basta T, Jensen ED, Klymkowsky MW. The beta-catenin/VegT-regulated early zygotic gene Xnr5 is a direct target of SOX3 regulation. Development. 2003;130(23):5609–24. Epub 2003/10/03. doi: 10.1242/dev.00798 . [DOI] [PubMed] [Google Scholar]
49.Li J, Pan G, Cui K, Liu Y, Xu S, Pei D. A dominant-negative form of mouse SOX2 induces trophectoderm differentiation and progressive polyploidy in mouse embryonic stem cells. J Biol Chem. 2007;282(27):19481–92. doi: 10.1074/jbc.M702056200 . [DOI] [PubMed] [Google Scholar]
50.Seo E, Basu-Roy U, Zavadil J, Basilico C, Mansukhani A. Distinct functions of Sox2 control self-renewal and differentiation in the osteoblast lineage. Mol Cell Biol. 2011;31(22):4593–608. Epub 2011/09/19. doi: 10.1128/MCB.05798-11 ; PubMed Central PMCID: PMC3209254. [DOI] [PMC free article] [PubMed] [Google Scholar]
51.Vickers ER, Sharrocks AD. The use of inducible engrailed fusion proteins to study the cellular functions of eukaryotic transcription factors. Methods. 2002;26(3):270–80. doi: 10.1016/S1046-2023(02)00031-2 . [DOI] [PubMed] [Google Scholar]
52.Ross S, Best JL, Zon LI, Gill G. SUMO-1 modification represses Sp3 transcriptional activation and modulates its subnuclear localization. Mol Cell. 2002;10(4):831–42. doi: 10.1016/s1097-2765(02)00682-2 . [DOI] [PubMed] [Google Scholar]
53.Holmstrom S, Van Antwerp ME, Iñiguez-Lluhi JA. Direct and distinguishable inhibitory roles for SUMO isoforms in the control of transcriptional synergy. Proc Natl Acad Sci U S A. 2003;100(26):15758–63. Epub 2003/12/08. doi: 10.1073/pnas.2136933100 ; PubMed Central PMCID: PMC307641. [DOI] [PMC free article] [PubMed] [Google Scholar]
54.Long J, Wang G, He D, Liu F. Repression of Smad4 transcriptional activity by SUMO modification. Biochem J. 2004;379(Pt 1):23–9. doi: 10.1042/BJ20031867 ; PubMed Central PMCID: PMC1224064. [DOI] [PMC free article] [PubMed] [Google Scholar]
55.Wu F, Zhang J, Wang P, Ye X, Jung K, Bone KM, et al. Identification of two novel phenotypically distinct breast cancer cell subsets based on Sox2 transcription activity. Cell Signal. 2012;24(11):1989–98. doi: 10.1016/j.cellsig.2012.07.008 . [DOI] [PubMed] [Google Scholar]
56.Wu Y, Guo Z, Wu H, Wang X, Yang L, Shi X, et al. SUMOylation represses Nanog expression via modulating transcription factors Oct4 and Sox2. PLoS One. 2012;7(6):e39606. Epub 2012/06/30. doi: 10.1371/journal.pone.0039606 ; PubMed Central PMCID: PMC3382131. [DOI] [PMC free article] [PubMed] [Google Scholar]
57.Chymkowitch P, Nguea PA, Enserink JM. SUMO-regulated transcription: challenging the dogma. Bioessays. 2015;37(10):1095–105. doi: 10.1002/bies.201500065 . [DOI] [PubMed] [Google Scholar]
58.Hay RT. SUMO: a history of modification. Mol Cell. 2005;18(1):1–12. doi: 10.1016/j.molcel.2005.03.012 . [DOI] [PubMed] [Google Scholar]
59.Yau TY, Molina O, Courey AJ. SUMOylation in development and neurodegeneration. Development. 2020;147(6). Epub 20200318. doi: 10.1242/dev.175703 ; PubMed Central PMCID: PMC7097199. [DOI] [PMC free article] [PubMed] [Google Scholar]
60.Thiel G. How Sox2 maintains neural stem cell identity. The Biochemical journal. 2013;450(3):e1–2. Epub 2013/03/01. doi: 10.1042/BJ20130176 . [DOI] [PubMed] [Google Scholar]
61.Liebelt F, Vertegaal AC. Ubiquitin-dependent and independent roles of SUMO in proteostasis. Am J Physiol Cell Physiol. 2016;311(2):C284–96. Epub 2016/06/24. doi: 10.1152/ajpcell.00091.2016 ; PubMed Central PMCID: PMC5129774. [DOI] [PMC free article] [PubMed] [Google Scholar]
62.Qi Y, Liu H, Lin X. Sumoylation Stabilizes Smoothened to Promote Hedgehog Signaling. Dev Cell. 2016;39(4):385–7. Epub 2016/11/23. doi: 10.1016/j.devcel.2016.11.001 . [DOI] [PubMed] [Google Scholar]
63.Wen D, Wu J, Wang L, Fu Z. SUMOylation Promotes Nuclear Import and Stabilization of Polo-like Kinase 1 to Support Its Mitotic Function. Cell reports. 2017;21(8):2147–59. Epub 2017/11/23. doi: 10.1016/j.celrep.2017.10.085 ; PubMed Central PMCID: PMC5728694. [DOI] [PMC free article] [PubMed] [Google Scholar]
64.Homem CC, Repic M, Knoblich JA. Proliferation control in neural stem and progenitor cells. Nat Rev Neurosci. 2015;16(11):647–59. Epub 2015/10/01. doi: 10.1038/nrn4021 ; PubMed Central PMCID: PMC4667397. [DOI] [PMC free article] [PubMed] [Google Scholar]
65.Cossec JC, Theurillat I, Chica C, Bua Aguin S, Gaume X, Andrieux A, et al. SUMO Safeguards Somatic and Pluripotent Cell Identities by Enforcing Distinct Chromatin States. Cell stem cell. 2018;23(5):742–57 e8. Epub 2018/11/08. doi: 10.1016/j.stem.2018.10.001 . [DOI] [PubMed] [Google Scholar]

PLoS One. doi: 10.1371/journal.pone.0298818.r001

Decision Letter 0

Anujith Kumar

25 May 2023

PONE-D-23-11587SUMO-dependent Transcriptional Repression by Sox2 Inhibits the Proliferation of Neural Stem CellsPLOS ONE

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Reviewer #1: Manuscript Number: PONE-D-23-11587

Manuscript Title: SUMO-dependent Transcriptional Repression by Sox2 Inhibits the Proliferation of Neural Stem Cells

Marelli et al. describe a study of roles of SUMOylation in the transcriptional activity of Sox2 in a human cell line of early neural progenitors. By transfecting a variety of cDNA constructs, including wild type Sox2, or a mutant Sox2 unable to be SUMOylated , or the same mutant “rescued” by C-Terminal addition of SUMO peptides, they demonstrate the ability of the rescued SUMOylated mutant to affect gene transcription in the transfected cell line, by increasing or repressing the activities of almost a thousand genes. The non-SUMOylatable Sox2 cDNA is instead essentially devoid of such activity. Within the subset of repressed genes, they point to a significant enrichment of genes related to cell cycle, and suggest that the inhibition of these genes may be relevant to the control of the progenitor proliferation kinetics and their differentiation. The identification of these cell-cycle related genes as target of repression by Sox2 is potentially important.

Major Comment 1

The experiments are, in general, performed in a technically sound way, with appropriate controls and repetitions adequate for statistical analysis, and the data are therefore credible. However, I have some reservations on the strategy used by the authors, and on the interpretation of the results. In Figure 1, they show that a non-SUMOylatable mutant is more efficient than a wt-Sox2 or “rescued” non-SUMOylatable Sox2 mutant in driving transcription of a Luciferase construct having a Sox2-binding sequence in the promoter. Thus, lack of SUMOylation does not seem to impair the transcriptional activity of Sox2.

As this type of experiment does not allow to test for repression, they try a different strategy, that yields results in contrast with those described above, suggesting that Sox2 SUMOylation matters, both in activation and in repression of transcription. Unfortunately, the authors do not discuss this discrepancy and do not discuss their results in sufficient depth.

This experiment is unusual, and rather difficult to interpret. The authors do not provide some important details, and do not describe the logic of the experiment, and possible caveats, in depth. To evaluate the role of Sox2 SUMOylation they transfect a wild type Sox2 cDNA construct, a mutant non-SUMOylatable Sox2 construct, and a rescued mutant non-SUMOylatable Sox2 carrying SUMOpeptides at its C-terminal (from now-on, wt Sox2, mut Sox2 and rescued mut Sox2, respectively).The recipient cell line is a human neural progenitor line. Importantly, neural progenitor lines typically express Sox2 at relatively high levels, meaning that gene expression in these cells reflects the action of Sox2, that controls a significant proportion of the genes. It is not clear to me how much Sox2 is expressed in these cells. If Sox2 is indeed present in these cells, significantly modifying the expression of genes regulated by ENDOGENOUS Sox2 may require the addition of large amounts of EXOGENOUS Sox2, in quantities that may well exceed physiological levels. In their transfection experiments (Fig.3), the authors show that both wt Sox2 and the rescued mut Sox2

(Sox2_K247R_SUMO2) are able to up- and down-regulate a small proportion of the expressed genes. In contrast, non-SUMOylatable mut Sox2 (Sox2_K247R9) is essentially devoid of both activating and repressing activity. The authors comment (lines 332-333):” This evidence clearly shows that SUMOylation of Sox2 has a strong repressive effect on target genes and that this effect is rescued by the absence of SUMOylation”. I do not agree with this statement, for two reasons: First, a similar effect, although somewhat less strong, is also obtained with wt Sox2. It is true that wt Sox2 can be SUMOylated, after being newly synthesized following cDNA transfection, but the authors report that only about 5% of Sox2 is SUMOylated within the cell, see lines 507-508. So, it remains to be proven that non-SUMOylated Sox2 is inactive in gene repression.

The second objection is that it is logically incorrect to equate the proportion of genes downregulated by Sox2 overexpression to the absolute activity of Sox2 expressed at physiological levels. Genes that are already downregulated by endogenous factors, including Sox2 itself, may well require much higher levels (not necessarily physiological) of Sox2 and other repressing factors to be further repressed. So, the different results obtained using mut Sox2 (Sox2_K247R9) versus rescued mut Sox2 (Sox2_K247R_SUMO2) might simply indicate that mutSox2 is somewhat less efficient in inducing a high level of repression than rescued mut Sox2, and fails to reach the high expression threshold necessary for further repression of endogenously repressed genes. This does not imply that the basic repression activity of Sox2 is altered or abolished.Note that, at lines 474-475 the authors report that mutSox2 is much less present in chromatin than rescued mut Sox2, which might contribute to the inability of mut Sox2 to further repress the expression of genes expressed in the transfected cells. These results, overall, do not imply that non-SUMOylated Sox2 is unable to repress Sox2 targets.

As to activation by SUMOylated versus non-SUMOylated Sox2, the authors suggest, again based on data in Fig.3, that only SUMOylated Sox2 is transcriptionally active (see lines 507-508). The reasoning reported above as to repression data, can be applied equally well to the activation data. Moreover, Fig.1 clearly shows that, when tested on the same reporter gene, carrying Sox2-binding sites, constitutively SUMOylated Sox2 is LESS active than wt Sox2 or non SUMOylatable Sox2

Major Comment 2

The genes analysed in Fig.3 represent the complete transcriptome of the tested cells. A proportion of these genes are presumably directly regulated by Sox2; in addition, genes regulated by Sox2 may encode factors which in turn affect the expression of genes non directly controlled by Sox2 ( for a knock-out experiment in mouse neural cells reporting expression data, see Bertolini J et al., 2019, Cell Stem Cell, PMID: 30849367). The authors should carefully consider these points in Discussion. Do transfected Sox2 constructs affect, in their experiments, only some of the physiological targets of Sox2? Overexpression experiments have the drawback that genes not regulated, in vivo, by the transcription factor under study, or only moderately affected by it, may be inappropriately activated; this effect is well known in molecular oncology. So the question is whether in their experiments the transfected constructs encoding Sox2 and its SUMOylatable or non SUMOylatable variants, activate/repress the real Sox2 targets. The authors might consult the data in Bertolini et al (see above) and ChIP-seqs for Sox2 available in the literature; see also D’Aurizio R.et al., 2022Int.J.Mol.Sci., PMID:35887306, who compare mouse with human data and provide many references to papers regarding ChIP-seqs).Defining which ones, among the repressed cell cycle-related genes, are indeed regulated in vivo by Sox2 would be very important.

Minor Comments

1)In the cell proliferation experiment of Fig.5, differences are initially detected between day 1 and 2, and slightly increase at day 3; then, curves increase in parallel for 2-3 days, when the curves start approaching a plateau. So, the effects of the transfected constructs seem to be only at the initial stages, then they progressively disappear.

2)lines 353-358

See major comment 1. Gene downregulation is observed with transfection of both wtSox2 and rescued mut Sox2 (Sox2_K247R_SUMO2.

3)lines507-509

I strongly disagree with this comment, see Major Comment 1

4)lines 516-520

“our hypothesis that 517 Sox2 SUMOylation regulates the protein activity as transcription factor” : this sentence is unclear.

“we showed that SUMOylated Sox2 acquires transcriptional 520 repression activity compared to non SUMOylatable mutant Sox2”: not true, see comment 1

5)lines 551-552 “Our data suggest that when Sox2 cannot be SUMOylated, it loses its ability to repress a set of cell-cycle regulating target 552 genes, resulting in a higher proliferation rate”. See comments 1 and 2. Also, the higher proliferation rate is very transient. The experiment shoul better be performed in long-term growing cell cultures.

6)It would important to have access to Tables listing the top 50 most upregulated and the top 50 most downregulated genes in the transfection experiments reported in Fig.3, together with their expression values, as Transcript per Million, and fold changes.

7) English spelling and some sentences should be revised, see for example “which we have showed affects (lines 383-4), “grew slower” (line 416), wells was analyzed (line 254), underling (line 561),and several other ones.

CONCLUSIONS

This paper presents some interesting results, in particular the observation of the downregulation of many genes, in particular genes involved in cell cycle control. If the authors can confirm that these downregulated genes are physiological Sox2 targets, shown to harbour Sox2 -binding sites, this is of great potential interest. As to the role of SUMOylation, at present the data are not entirely convincing, in my opinion. The ideal experiment would be to introduce a mutation within the SUMOylation site of Sox2 by CRISPR-CAS technology, and to select appropriate cell lines. I have to recognize, however, that this is a complex experiment. Alternatively, once a bona fide Sox2 target gene is identified among the repressed genes, one might transfect an appropriate construct into Sox2-null cells, and then transfect these cells with SUMOylated (or SUMOylatable) and non-SUMOylatable Sox2 constructs to study the relative effects of these constructs on this target.

Reviewer #2: In the present work, the authors studied the role of Sox2 SUMOylation in human neural stem cells (NSCs). They generated Sox2 mutants with diminished SUMOylatability and mutants with a SUMO fusion (constitutively SUMOylated) and studied their transcriptional activity on a reporter system and their effect on NSCs’ transcriptome and proliferation. They found that the variants fused to SUMO lost their transcriptional activation activity compared to the wt Sox2 and acquired transcriptional repression activity compared to non SUMOylatable Sox2 mutants. They identified a set of genes that were differentially regulated by the different Sox2 variants in NSCs. Interestingly, the genes repressed by Sox2 in a SUMO-dependent manner are associated with cell cycle control. In agreement, they also found that SUMOylation of Sox2 affected the proliferation of these cells, since the NSCs transfected with wt Sox2 or Sox2 fused to SUMO grew slower than control cells or that cells transfected with the Sox2 mutant with impaired SUMOylation. Since they did not detect differences in cell viability, they conclude that the effect is on the proliferation rate. Finally, the authors studied the subcellular localization of the different mutants through western blot analysis of subcellular fractions. Although they did not find any differences in their localization, they detected differences in the expression of the different mutants and proposed that SUMOylation may stabilize Sox2, but they suggest that this may not be the only role played by SUMOylation in the regulation of Sox2 activity. Finally, they commented that various genes differentially regulated by both wt Sox2 and the SUMO fusion are involved in human syndromes linked to microcephaly and suggested a link between the loss of activity of Sox3, a member of Sox family, and microcephaly.

Major points:

-Interference with the SUMOylation pathway, for example by transfection of a dominant negative mutant of the UBC9 enzyme, at least in the reporter assay, would strengthen the conclusions.

-The authors could complement the localization analysis with a microscopy approach, ideally in living cells expressing fusions of the different mutants to a fluorescent protein; or alternatively, by immunofluorescence of fixed cells (they use an Anti-Myc antibody to detect by Western blot the expressed constructions that have the Myc tag).

Minor points:

-The authors select transfected cells by FACS for the RNA-seq analysis; however, I could not find any description of selection or identification of transfected cells in the proliferation analysis. How much of the cell population is transfected? They mention that the differences observed “tended to disappear around 5-6 days after transfection, presumably due to degradation of the plasmid DNA transfected and its encoded protein.” In my opinion, identification of transfected cells (for example by expressing the Sox2 variants fused to a fluorescent protein) would strengthen the conclusions.

- Analysis of the stability of the different Sox2 variants would contribute to the discussion about the role played by SUMOylation in the regulation of Sox2 activity.

-At the end of the introduction section, a paragraph with the most relevant findings of the manuscript is missing.

-Tables 1 and 2 are the same.

- In my opinion, data from figures 5 and 6 should be included in one figure, it is not necessary to show the three replicates in the way that are presented in the main figure 6A, and scale bars should be included in Figure 6B.

-Line 254: Wells was analyzed should be replaced by Wells were analyzed.

-Line 453: fester should be replaced by faster.

-Line 487: maintaining their stemness state should be replaced by maintaining their stemness.

Reviewer #3: In this study, Marelli et al investigated how SOX2, SOX2 mutant deficient of SUMOylation, and SOX2-SUMO fusion proteins regulate transcription and cell proliferation in neural stem cells. They observed that SOX2 represses more genes than it activates. The genes repressed by SOX2 are associated with centrosomes, centromeres and other aspects of cell cycle control. In addition, they observed that the SOX2-SUMO fusion protein is more potent in repression as well as activation than wildtype SOX2, whereas the SUMOylation-defective mutant is poorly active in regulation of gene expression. Thus, they conclude that SUMOylation of Sox2 is necessary for its repression of gene expression and for its repressive effects on cell proliferation. However, there are a nubmer of issues needed to be addressed before I will support its publication.

1. In Fig. 1, authors showed that K247A and K247R mutants were as active as SOX2 in transcriptional activation of a reporter gene. The Western blot data for expression of various SOX2 mutants should be showed in this figure to interpret the luciferase reporter assay.

2. Since K247A and K247R were highly active in assay in Fig. 1, why it was essnetially inactive in transcriptional regulation (only repressed 19 and activated 11 genes) in neural stem cells?

3. A major concern for the study is that RNA-seq was performed for each type of SOX2 proteins for only one dose. Much of the differences in regulation of gene expression could be attributed to different levels of SOX2 expression. For instance, K247R mutant could be expressed very low in this experiment, and thus had little effect on gene expression. It would be more convincing if two or three differnt doses of SOX2 were tested for each and data were compared among the samples that have similar level of SOX2 proteins.

4. Authors need to address if SUMOylation influences SOX2 transcriptional activity primarily by affecting SOX2 protein stability or promoting its repression activity or both.

5. Introduction is too long and some of information could be deleted or moved to discussion.

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Reviewer #1: Yes: SERGIO OTTOLENGHI

Reviewer #2: No

Reviewer #3: No

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PLoS One. 2024 Mar 20;19(3):e0298818. doi: 10.1371/journal.pone.0298818.r002

Author response to Decision Letter 0

11 Dec 2023

Dear PlosONE editorial team,

Although, we are now no longer in a position to carry out more experiments since the Scotting lab was closed due to Dr. Scotting’s retirement, we have addressed all of the reviewers’ comments in our response below and in the edits that we have applied to the paper.

Reviewer 1, major comment 1: “The experiments are, in general, performed in a technically sound way, with appropriate controls and repetitions adequate for statistical analysis, and the data are therefore credible. However, I have some reservations on the strategy used by the authors, and on the interpretation of the results. In Figure 1, they show that a non-SUMOylatable mutant is more efficient than a wt-Sox2 or “rescued” non-SUMOylatable Sox2 mutant in driving transcription of a Luciferase construct having a Sox2-binding sequence in the promoter. Thus, lack of SUMOylation does not seem to impair the transcriptional activity of Sox2. As this type of experiment does not allow to test for repression, they try a different strategy, that yields results in contrast with those described above, suggesting that Sox2 SUMOylation matters, both in activation and in repression of transcription. Unfortunately, the authors do not discuss this discrepancy and do not discuss their results in sufficient depth.”

The data shown in figure 1 suggests that by blocking Sox2 SUMOylation, its transcriptional activation ability is increased compared to wild-type (wt) Sox2. Conversely, constitutive SUMOylation of Sox2 has the opposite effect of diminishing the protein’s ability to transcriptionally activate the reporter gene. This assay is not representative of an in vivo system, as the promoter controlling the expression of the Luciferase gene has been artificially designed by inserting three repetitions of a Sox2 binding motif in a generic promoter (pTl/3xSX promoter, as published by Liu et al. [1]). In vivo, Sox2 binding consensus motif can vary [2] and Sox2 can regulate target genes both directly and indirectly [3, 4]. Therefore, as stated in the manuscript’s results section, this assay only allowed us to detect and measure direct transcriptional activation of the artificial promoter and was used as a screening method to test several Sox2 mutant constructs to assess if the mutations applied impacted the protein’s transcriptional activity compared to wt Sox2. In order to make this clearer, we have added the following to the paper: “The luciferase Reporter Assay showed that SUMOylation appears to inhibit the activation activity of Sox2. However, this assay only assessed the ability of Sox2 to activate expression driven by one particular artificial promoter in HeLa cells and did not assess transcriptional repression. Therefore, the effects of SUMOylation on Sox2 transcriptional activity were further analysed using a genome-wide approach in hNSCs” (lines 350-355).

Moreover, while our study focussed mainly on the effects of Sox2 SUMOylation on its transcriptional repression ability, we do not intend to imply that Sox2 transcriptional activation ability is not also affected by SUMOylation. In fact, the RNA sequencing data shown in figure 3 suggests that this is also the case. We decided to focus our study on the effect on transcriptional repression for the following reasons. Many genes repressed by exogenous wt Sox2 and SUMOylated sox2 were common to both constructs while gene activated were not; the effect of loss of SUMOylation of Sox2 affected repressed genes more strongly than activated genes; only those genes repressed fell into functional groups and many of these were cell cycle related, which has a direct link with the effect of exogenous Sox2 on cell proliferation.

Reviewer 1, major comment 1: “To evaluate the role of Sox2 SUMOylation they transfect a wild type Sox2 cDNA construct, a mutant non-SUMOylatable Sox2 construct, and a rescued mutant non-SUMOylatable Sox2 carrying SUMO peptides at its C-terminal (from now-on, wt Sox2, mut Sox2 and rescued mut Sox2, respectively). The recipient cell line is a human neural progenitor line. Importantly, neural progenitor lines typically express Sox2 at relatively high levels, meaning that gene expression in these cells reflects the action of Sox2, that controls a significant proportion of the genes. It is not clear to me how much Sox2 is expressed in these cells. If Sox2 is indeed present in these cells, significantly modifying the expression of genes regulated by ENDOGENOUS Sox2 may require the addition of large amounts of EXOGENOUS Sox2, in quantities that may well exceed physiological levels.”

We acknowledge that levels of expressions of endogenous and exogenous Sox2 might play an important role in the interpretation of our RNA sequencing data. Therefore, we have added further data in the supplementary (Supplementary figure S3): the western blot below, which was performed for a different purpose, was initially probed with anti-Myc primary antibody (exogenous Sox2 is Myc tagged) and subsequently with anti-Sox2 primary antibody. The blot shows bands of comparable brightness between endogenous and exogenous Sox2. Although these are different primary antibodies and might have slightly different avidity, we used the same fluorescein-tagged anti-mouse secondary antibody to visualize them. We believe it is reasonable to conclude that the levels of exogenous Sox2 are not dissimilar to the levels of endogenous Sox2. Therefore, we have added the following statement in the manuscript: “The expression levels of the exogenous constructs achieved with our transfection protocol was comparable to the levels of endogenous Sox2 (S5 Fig)” (lines 376-378. This is consistent with the new data we present below, showing that many of the Sox2 targets we have identified, have also been identified through ChIP-Seq analysis in other published studies. Reviewer 1, major comment 1: “In their transfection experiments (Fig.3), the authors show that both wt Sox2 and the rescued mut Sox2 (Sox2_K247R_SUMO2) are able to up- and down-regulate a small proportion of the expressed genes. In contrast, non-SUMOylatable mut Sox2 (Sox2_K247R9) is essentially devoid of both activating and repressing activity. The authors comment (lines 332-333):” This evidence clearly shows that SUMOylation of Sox2 has a strong repressive effect on target genes and that this effect is rescued by the absence of SUMOylation”. I do not agree with this statement, for two reasons: First, a similar effect, although somewhat less strong, is also obtained with wt Sox2. It is true that wt Sox2 can be SUMOylated, after being newly synthesized following cDNA transfection, but the authors report that only about 5% of Sox2 is SUMOylated within the cell, see lines 507-508. So, it remains to be proven that non-SUMOylated Sox2 is inactive in gene repression.”

Since the Sox2_K247R mutant construct loses the ability to affect the genes that are repressed by wt Sox2, this implies that the effect of exogenous wt Sox2 on these genes is due to that proportion of exogenous Sox2 that becomes SUMOylated. Hence, the reason why the rescued mutant construct (Sox2_K247R-SUMO2) has a stronger transcriptional regulation effect than the wt, is because 100% of the protein produced is now SUMOylated. Together, these data imply that the effects of the expression of these constructs on the genes identified is predominantly due to the SUMOylation of Sox2.

If non-SUMOylated Sox2 were in general active as a repressor, we would expect a much greater number of the identified genes to be repressed by exogenous, non-SUMOylatable Sox2 (Sox2_K247R).

The hypothesis that the portion of exogenous wt Sox2 that becomes SUMOylated is responsible for the transcriptional regulation of a high number of genes, is consistent with the published literature. Yau et al. published that: “SUMOylation targets are rarely quantitatively SUMOylated. In fact, in the case of most targets, only a small percentage of a SUMO target protein is SUMOylated at any given time. This is paradoxical because, as will become clear, SUMOylation often modulates the activity of nearly the entire population of a protein target. This phenomenon has been termed the ‘SUMO enigma’” [5, 6].

In order to clarify this point, we have added the following:

“Since the Sox2_K247R mutant construct loses the ability to affect the genes that are repressed by wt Sox2, this implies that the effect seen upon expression of exogenous wt Sox2 on these genes is due to that proportion of exogenous Sox2 that becomes SUMOylated. Hence, the reason why the rescued mutant construct (Sox2_K247R-SUMO2) has a stronger transcriptional regulation effect than the wt Sox2, is likely to be because all of the protein produced is now SUMOylated. Together, these data imply that the effects of the expression of these constructs on the genes identified is predominantly due to the SUMOylation of Sox2. This is consistent with the published literature which reports that, despite a small proportion of SUMO target proteins being actually SUMOylated, it is often that SUMOylated proportion of the protein that regulates most of the transcriptional activity of the protein. This phenomenon has been termed the ‘SUMO enigma’ [5, 6]” (lines 404-414).

Reviewer 1, major comment 1: “The second objection is that it is logically incorrect to equate the proportion of genes downregulated by Sox2 overexpression to the absolute activity of Sox2 expressed at physiological levels. Genes that are already downregulated by endogenous factors, including Sox2 itself, may well require much higher levels (not necessarily physiological) of Sox2 and other repressing factors to be further repressed. So, the different results obtained using mut Sox2 (Sox2_K247R9) versus rescued mut Sox2 (Sox2_K247R_SUMO2) might simply indicate that mutSox2 is somewhat less efficient in inducing a high level of repression than rescued mut Sox2 and fails to reach the high expression threshold necessary for further repression of endogenously repressed genes. This does not imply that the basic repression activity of Sox2 is altered or abolished. Note that, at lines 474-475 the authors report that mutSox2 is much less present in chromatin than rescued mut Sox2, which might contribute to the inability of mut Sox2 to further repress the expression of genes expressed in the transfected cells. These results, overall, do not imply that non-SUMOylated Sox2 is unable to repress Sox2 targets.”

Although we accept that we cannot make any certain predictions of the role of SUMOylation of the endogenous Sox2, we would argue that the strength of the effect caused by loss of SUMOylation of the exogenous Sox2, suggests that SUMOylation of endogenous Sox2 is likely to have a substantial effect on its ability to repress target genes. So, in the paper, we have now re-phrased our statement for the interpretation of the data. Rather than “This evidence clearly shows that SUMOylation of Sox2 has a strong repressive effect on target genes and that this effect is rescued by the absence of SUMOylation”, we now say: “Since our experiments relied on the function of exogenous Sox2 constructs we compared the genes affected to those identified as targets of endogenous Sox2 via Chip-Seq analysis. Comparison to the published studies in mouse ESCs and NSCs revealed that 97 of the 165 genes downregulated in our studies were identified as direct Sox2 targets using Chip-Seq analysis. Together with our observation that the levels of exogenous Sox2 appears to be comparable to the level of endogenous Sox2 (Figure S3), this suggests that, although it’s possible that the endogenous Sox2 could affect some non-physiological targets, SUMOylation of endogenous Sox2 is also likely to play a role in its ability to repress target genes.”

“Since our experiments relied on the function of exogenous Sox2 constructs, we compared the genes affected to those identified as targets of endogenous Sox2 via Chip-Seq analysis. Comparison to the published studies in mouse ESCs and NSCs revealed that 97 of the 165 genes downregulated in our studies were identified as direct Sox2 targets using Chip-Seq analysis. Together with our observation that the levels of exogenous Sox2 appears to be comparable to the level of endogenous Sox2 (Figure S3), this suggests that, although it’s possible that the endogenous Sox2 could affect some non-physiological targets, SUMOylation of endogenous Sox2 is also likely to play a role in its ability to repress target genes” (lines 420-427).

Reviewer 1, major comment 1: “As to activation by SUMOylated versus non-SUMOylated Sox2, the authors suggest, again based on data in Fig.3, that only SUMOylated Sox2 is transcriptionally active (see lines 507-508). The reasoning reported above as to repression data, can be applied equally well to the activation data. Moreover, Fig.1 clearly shows that, when tested on the same reporter gene, carrying Sox2-binding sites, constitutively SUMOylated Sox2 is LESS active than wt Sox2 or non SUMOylatable Sox2.”

As mentioned above, we have now included more details on the difference between the luciferase and RNA seq data.

Reviewer 1 concludes the major comment 1 by stating that “Fig.1 clearly shows that, when tested on the same reporter gene, carrying Sox2-binding sites, constitutively SUMOylated Sox2 is LESS active than wt Sox2 or non SUMOylatable Sox2”. It is important to point out that the data presented in figure 1 only show that SUMOylated Sox2 is less active in activating the reporter gene. As stated, this assay does not give an indication of the repression ability of the constructs. This is consistent with RNA sequencing data, which shows that SUMOylated Sox2 has a major impact on the number of genes that are down-regulated. It also has a lesser impact on genes that are up-regulated, but that do not share a particular known physiological function.

Reviewer 1, major comment 2: “The genes analysed in Fig.3 represent the complete transcriptome of the tested cells. A proportion of these genes are presumably directly regulated by Sox2; in addition, genes regulated by Sox2 may encode factors which in turn affect the expression of genes non directly controlled by Sox2 (for a knock-out experiment in mouse neural cells reporting expression data, see Bertolini J et al., 2019, Cell Stem Cell, PMID: 30849367). The authors should carefully consider these points in Discussion. Do transfected Sox2 constructs affect, in their experiments, only some of the physiological targets of Sox2? Overexpression experiments have the drawback that genes not regulated, in vivo, by the transcription factor under study, or only moderately affected by it, may be inappropriately activated; this effect is well known in molecular oncology. So, the question is whether in their experiments the transfected constructs encoding Sox2 and its SUMOylatable or non SUMOylatable variants, activate/repress the real Sox2 targets. The authors might consult the data in Bertolini et al (see above) and ChIP-Seq for Sox2 available in the literature; see also D’Aurizio R.et al., 2022Int.J.Mol.Sci., PMID:35887306, who compare mouse with human data and provide many references to papers regarding ChIP-Seq).Defining which ones, among the repressed cell cycle-related genes, are indeed regulated in vivo by Sox2 would be very important.”

We think this is a valid point and we would like to thank reviewer 1 for highlighting this. We have now compared our RNA sequencing data with published ChIP sequencing data and added the results of this analysis to the paper. We are pleased to report that this confirms that a high proportion of the genes we identified as repressed by both exogenous wt Sox2 and Sox_K247R-SUMO2, and in particular cell cycle related genes, have indeed been identified in these ChIP-Seq data. The following sections have been added to the paper:

“By comparing the effects of over-expressing exogenous wt Sox2, non-SUMOylatable Sox2 mutant or constitutively SUMOylated Sox2 in human neural stem cells (hNSC), we found that SUMOylated Sox2, similarly to wt (SUMOylatable) Sox2, caused the downregulation of a set of proliferation related genes. Many of these genes have been published as Sox2 targets in Chromatin Immunoprecipitation Sequencing (ChIP-Seq) assay” (lines 119-123).

“In the same comparison with published Chip-Seq data used above, 22 of the 32 cell cycle-related genes downregulated in our studies were identified as direct Sox2 targets” (lines 462-464).

In addition, Maresca et al. [4] investigate Sox2 activity as a pioneering transcription factor. They show that Sox2 binding keeps the chromatin in an open state which allows other transcription factor to bind to their target genes. This could explain why some of the genes that we identified as differentially regulated are not found in ChIP sequencing data from literature. These genes could be genes that are indirectly regulated by Sox2 as a pioneering transcription factor: they don’t have a Sox2 binding site because they are not directly regulated by Sox2.

In response to reviewer 1 minor comments:

1) “In the cell proliferation experiment of Fig.5, differences are initially detected between day 1 and 2, and slightly increase at day 3; then, curves increase in parallel for 2-3 days, when the curves start approaching a plateau. So, the effects of the transfected constructs seem to be only at the initial stages, then they progressively disappear”.

As stated in the manuscript, this is due to the cells reaching maximum confluency and therefore not having any space left to grow further. It is also visible in Figure 6b, which shows completely confluent cells at day 7. Moreover, the transfected plasmid and encoded protein are susceptible to degradation over time and therefore it is expected that their effect would gradually decrease over the course of days. To deliver this concept in a clearer manner, we have amended the manuscript as follows: “Statistical comparison between the proliferation rates of the 4 samples showed that the proliferation rate of cells over-expressing either wt Sox2 or Sox2_K247R-SUMO2 was significantly lower than the negative control (pcDNA3) at days 2, 3 and 4 (Figure 6A). These differences tended to disappear around 5-6 days after transfection, presumably due to degradation of the transfected plasmid DNA transfected and its encoded protein and to the cells reaching complete confluency. At day 7 after transfection, there was a slight decrease in the proliferation detected, presumably due to cell death and cell detachment caused by high cell confluency (Figure 6B, panel d7)” (lines 497-503).

2) “See major comment 1. Gene downregulation is observed with transfection of both wtSox2 and rescued mut Sox2 (Sox2_K247R_SUMO2).”

We are not clear what the reviewer is asking with this comment. Please see our response to major comment 1.

3) “Lines 507-509 I strongly disagree with this comment, See Major Comment 1”.

We have addressed major comment 1 above.

4) “Lines 516-520 ‘our hypothesis that Sox2 SUMOylation regulates the protein activity as a transcription factor’: this sentence is unclear. ‘We showed that SUMOylated Sox2 acquires transcriptional repression activity compared to non SUMOylatable mutant Sox2’: not true, see comment 1”.

We agree that that sentence is unclear and did add little to our discussion, repeating points made elsewhere, so we have removed it.

5) “Lines 551-552 “Our data suggest that when Sox2 cannot be SUMOylated, it loses its ability to repress a set of cell-cycle regulating target 552 genes, resulting in a higher proliferation rate”. See comments 1 and 2. Also, the higher proliferation rate is very transient. The experiment should better be performed in long-term growing cell cultures”.

Again, this statement repeats what was said elsewhere so it has been removed.

6) “It would be important to have access to Tables listing the top 50 most upregulated and the top 50 most downregulated genes in the transfection experiments reported in Fig.3, together with their expression values, as Transcript per Million, and fold changes”.

We acknowledge that this is a valid point and we have now added these data to the supplementary data.

Reviewer 2, major point 1: “interference with the SUMOylation pathway, for example by transfection of a dominant negative mutant of the UBC9 enzyme, at least in the reporter assay, would strengthen the conclusions”.

We have performed some preliminary experiments looking and SUMOylation pathway and in particular co-transfecting Ubc9 together with wild-type Sox2 to enhance SUMOylation. However, this did not lead to a detectable increase in endogenously SUMOylated Sox2, probably because SUMOylation is tightly regulated and over-expression of Ubc9 might not be enough to bypass such regulation. For this reason, we designed our experimental approach including constitutively SUMOylated mutant Sox2 constructs. Unfortunately, we are currently not able to perform additional experiments due to closure of the laboratory following Dr. Scotting’s retirement.

Reviewer 2, major point 2: “The authors could complement the localization analysis with a microscopy approach, ideally in living cells expressing fusions of the different mutants to a fluorescent protein; or alternatively, by immunofluorescence of fixed cells (they use an Anti-Myc antibody to detect by Western blot the expressed constructions that have the Myc tag)”.

The suggestion of investigating Sox2/SUMOylated Sox2 localisation through microscopy is indeed appealing. However, we do not currently have possibility undertake this. We believe that our data is of great potential interest for other research groups studying SUMOylation and we agree that it would be very interesting to see if differences in localisation between SUMOylated and non SUMOylated Sox2 can be detected by microscopy.

Reviewer 2, minor point 1: “The authors select transfected cells by FACS for the RNA-seq analysis; however, I could not find any description of selection or identification of transfected cells in the proliferation analysis. How much of the cell population is transfected? They mention that the differences observed ‘tended to disappear around 5-6 days after transfection, presumably due to degradation of the plasmid DNA transfected and its encoded protein.’ In my opinion, identification of transfected cells (for example by expressing the Sox2 variants fused to a fluorescent protein) would strengthen the conclusions.”

Reviewer 2 raises an interesting suggestion regarding the proliferation experiment. Unfortunately, this assay does not allow for expression of fluorescent proteins, as that would interfere with the experimental read-out, which is made by reading the emissions of AlamarBlueTM Cell Viability Reagent. We have performed transfections numerous times on this cell lines using the protocol adopted and we have always reached similar efficiency levels. In addition, the proliferation experiment was repeated three times (three biological replicates) and within each replicate, the experiment was performed on three independent wells for each construct at each time point (three technical replicates within each biological replicate). Therefore, this would compensate for any small change in transfection efficiency between experiments.

As reviewer 2 suggests, we are adding a paragraph with the most relevant findings at the end of the introduction section.

-Reviewer 2: “Tables 1 and 2 are the same.”

We have now amended these tables.

Reviewer 2, minor point 5: “In my opinion, data from figures 5 and 6 should be included in one figure, it is not necessary to show the three replicates in the way that are presented in the main figure 6A, and scale bars should be included in Figure 6B”.

We have amended the picture including only averages of the three biological replicates in Figure 6 and added scale bars to Figure 6A.

Reviewer 3, major point 1: “In Fig. 1, authors showed that K247A and K247R mutants were as active as SOX2 in transcriptional activation of a reporter gene. The Western blot data for expression of various SOX2 mutants should be showed in this figure to interpret the luciferase reporter assay”.

In response to reviewer 3, the Luciferase assay presented in figure 1 was repeated 3 times (3 biological replicates), with 4 technical replicas for each biological replicate, therefore we believe that the results are solid. We are including a western blot (shown below) showing expression of the transfected Sox2 mutant constructs in the supplementary data (Figure S1). This western blot was performed as part of a separate experiment, but we believe is a good indication of successful expression of the Sox2 constructs transfected. Reviewer 3, major point 2: “Since K247A and K247R were highly active in assay in Fig. 1, why it was essentially inactive in transcriptional regulation (only repressed 19 and activated 11 genes) in neural stem cells?”

As stated above in response to reviewer’s 1 major comment, the luciferase activation assay is designed based on a single artificial promoter containing three Sox2 binding sites and can only measure direct activation of this single, artificial promoter in HeLa cells. While this assay provides a good indication to investigate if there are differences in the transcriptional activity of Sox2 mutant constructs compared to the wt protein, it does not show how the protein performs in regulating target genes physiologically. As mentioned above in response to reviewer 1, we have now added the following statement to the paper to make this clearer: “the luciferase reporter assay showed that SUMOylation appears to inhibit the activation activity of Sox2. However, this assay only assessed the ability of Sox2 to activate expression when driven by one particular artificial promoter in HeLa cells and did not assess transcriptional repression. Therefore, the effects of SUMOylation on Sox2 transcriptional activity were further analysed using a genome-wide approach in hNSCs” (lines 350-355).

Reviewer 3, major point 3: “A major concern for the study is that RNA-seq was performed for each type of SOX2 proteins for only one dose. Much of the differences in regulation of gene expression could be attributed to different levels of SOX2 expression. For instance, K247R mutant could be expressed very low in this experiment, and thus had little effect on gene expression. It would be more convincing if two or three different doses of SOX2 were tested for each and data were compared among the samples that have similar level of SOX2 proteins”.

We agree that this could be a concern, however expression of the transfected proteins was assessed through western blot (supplementary figure S1). Unfortunately, repeating this experiment multiple times with different doses of DNA transfected was not doable due to the cost of RNA sequencing. Importantly, we want to point out that the RNA sequencing experiment was repeated three times for each Sox2 construct (three independent biological replicates), therefore generating a total of 12 samples (three vector only, three wild-type Sox2, three Sox2_K472R and three Sox2_K247R-SUMO2) and that the differentially expressed genes shown in figure 3 were present in all three replicates. We apologise if this was not clear in the manuscript, we have added the following statement in the results section: “The experiment was repeated three times (three biological replicates) for each condition (transfection of empty vector, wt Sox2, Sox2_K247R, Sox2_K247R-SUMO2) generating 12 samples, which were analysed by RNA sequencing” (lines 378-380).

Reviewer 3, major point 4: “Authors need to address if SUMOylation influences SOX2 transcriptional activity primarily by affecting SOX2 protein stability or promoting its repression activity or both”.

Reviewer 3 raises an important point in comment 4. Western blot analysis (figure S1) suggests that there is not a major difference in the expression levels of exogenous wt Sox2 compared to Sox2_K247R and therefore the dramatic difference in transcriptional activity seen both via Luciferase assay and via RNA sequencing is likely due to a difference in activity between these two constructs. The only difference between these two constructs is the point mutation (K247R) that abolishes SUMOylation and therefore we can conclude that the differential transcriptional activity is likely due to SUMOylation. By close inspection of figure S1, it might appear that Sox2_K247R-SUMO2 is expressed at slightly lower levels compared to wild-type Sox2 and Sox2_K247R. This is not clearly evident, especially because of the difference in size between the constructs, which run differently on an SDS-PAGE. However, if this was true, it would be in line with our hypothesis, as despite being expressed at lower level, this construct has a dramatic effect on the regulation of target genes, indicating that indeed SUMOylation plays a pivotal role in regulating Sox2 transcriptional activity. On the other hand, our subcellular localisation data show that Sox2-SUMO2 fusion proteins (Sox2-SUMO2 and Sox2_K247R-SUMO2) are present in the cells at slightly higher levels compared to wild-type Sox2 and Sox2_K247R. This could potentially suggest that SUMOylation stabilises Sox2. However, this difference is not statistically significant. Therefore, we cannot determine whether SUMOylation has an effect on Sox2 protein stability based on the data presented in our study. Indeed, this is a very interesting question that would require more sensitive assays to find a definitive answer.

Reviewer 3, major point 5: “Introduction is too long and some of information could be deleted or moved to discussion.”

We have addressed this by editing the original introduction to make it over 20% shorter and more focussed. However, as suggested by this referee we have then added a section with the most relevant findings at the end, bringing the final length to 85% of the original.

Please note, original uncropped and unadjusted images underlying all blot results are included in Supporting Information.

In conclusion, we believe that the identification of Sox2 target genes involved in the regulation of neural stem cells proliferation is of great interest. Moreover, by showing the involvement of SUMOylation in the regulation Sox2 transcriptional activity, we uncover a novel mechanism that has not been previously investigated in this context.

References

1. Liu YR, Laghari ZA, Novoa CA, Hughes J, Webster JR, Goodwin PE, et al. Sox2 acts as a transcriptional repressor in neural stem cells. BMC Neurosci. 2014;15:95. doi: 10.1186/1471-2202-15-95. PubMed PMID: 25103589; PubMed Central PMCID: PMC4148960.

2. Yesudhas D, Anwar MA, Panneerselvam S, Kim HK, Choi S. Evaluation of Sox2 binding affinities for distinct DNA patterns using steered molecular dynamics simulation. FEBS Open Bio. 2017;7(11):1750-67. Epub 20171009. doi: 10.1002/2211-5463.12316. PubMed PMID: 29123983; PubMed Central PMCID: PMCPMC5666385.

3. Holmes ZE, Hamilton DJ, Hwang T, Parsonnet NV, Rinn JL, Wuttke DS, et al. The Sox2 transcription factor binds RNA. Nat Commun. 2020;11(1):1805. Epub 20200414. doi: 10.1038/s41467-020-15571-8. PubMed PMID: 32286318; PubMed Central PMCID: PMCPMC7156710.

4. Maresca M, van den Brand T, Li H, Teunissen H, Davies J, de Wit E. Pioneer activity distinguishes activating from non-activating SOX2 binding sites. EMBO J. 2023:e113150. Epub 20230911. doi: 10.15252/embj.2022113150. PubMed PMID: 37691488.

5. Hay RT. SUMO: a history of modification. Mol Cell. 2005;18(1):1-12. doi: 10.1016/j.molcel.2005.03.012. PubMed PMID: 15808504.

6. Yau TY, Molina O, Courey AJ. SUMOylation in development and neurodegeneration. Development. 2020;147(6). Epub 20200318. doi: 10.1242/dev.175703. PubMed PMID: 32188601; PubMed Central PMCID: PMCPMC7097199.

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PLoS One. doi: 10.1371/journal.pone.0298818.r003

Decision Letter 1

Anujith Kumar

31 Jan 2024

SUMO-dependent Transcriptional Repression by Sox2 Inhibits the Proliferation of Neural Stem Cells

PONE-D-23-11587R1

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PLoS One. doi: 10.1371/journal.pone.0298818.r004

Acceptance letter

Anujith Kumar

22 Feb 2024

PONE-D-23-11587R1

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Supplementary Materials

S1 Fig. Visual assessment of apoptosis assay performed on hNSC grown in the presence of G418 (positive controls) or transfected with different Sox2 constructs and cultured O/N.

Scale bars: 50 μm.

(TIFF)

pone.0298818.s001.tiff^{(3.5MB, tiff)}

S2 Fig. Measurements of apoptosis on day 1 sets of wells incubated for different amounts of time after addition of CellEvent.

Two biological replicates were performed. Within each biological replicates, two technical replicates were performed. Bars indicate standard deviation of the mean.

(TIFF)

pone.0298818.s002.tiff^{(385KB, tiff)}

S3 Fig. Expression of Sox2 constructs in HeLa cells.

Membrane was probed using anti-Myc primary antibody followed by green Licor secondary antibodies.

(TIFF)

pone.0298818.s003.tiff^{(1.4MB, tiff)}

S4 Fig. Western blot performed on hNSC to assess the expression of Sox2 constructs used for RNA Sequencing.

Anti-Myc antibody was used to detect exogenous Sox2 proteins.

(TIFF)

pone.0298818.s004.tiff^{(452.2KB, tiff)}

S5 Fig. Western blot performed on lysates of hNSC transfected with wt Sox2 or Sox_K247R or co-transfected with wt Sox2 or Sox2_K247R, SUMO2 and Ubc9.

(TIFF)

pone.0298818.s005.tiff^{(1.8MB, tiff)}

S6 Fig. Proliferation of hNSC after transient transfection with either pcDNA3, wt Sox2, Sox2_K247R or Sox2_K247R-SUMO2 and incubation with Alamar Blue for 4 hours.

Bars indicate standard error of the mean.

(TIFF)

pone.0298818.s006.tiff^{(335.5KB, tiff)}

S1 Table. Genes differentially expressed by wt Sox2 and Sox2_K247R-SUMO2 associated with human microcephaly.

(TIF)

pone.0298818.s007.tif^{(451.7KB, tif)}

S1 File. Minimal data sets underlying the results presented and original western blot images.

(XLSX)

pone.0298818.s008.xlsx^{(2.4MB, xlsx)}

Attachment

Submitted filename: Response to Reviewers.docx

pone.0298818.s009.docx^{(307.8KB, docx)}

Data Availability Statement

All relevant data are within the paper and its Supporting Information files.

[pone.0298818.ref001] 1.Laudet V, Stehelin D, Clevers H. Ancestry and diversity of the HMG box superfamily. Nucleic Acids Res. 1993;21(10):2493–501. Epub 1993/05/25. doi: 10.1093/nar/21.10.2493 ; PubMed Central PMCID: PMC309552. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0298818.ref002] 2.Harley VR, Lovell-Badge R, Goodfellow PN. Definition of a consensus DNA binding site for SRY. Nucleic Acids Res. 1994;22(8):1500–1. Epub 1994/04/25. doi: 10.1093/nar/22.8.1500 ; PubMed Central PMCID: PMC308012. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0298818.ref003] 3.Ferrari S, Harley VR, Pontiggia A, Goodfellow PN, Lovell-Badge R, Bianchi ME. SRY, like HMG1, recognizes sharp angles in DNA. EMBO J. 1992;11(12):4497–506. Epub 1992/12/01. doi: 10.1002/j.1460-2075.1992.tb05551.x ; PubMed Central PMCID: PMC557025. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0298818.ref004] 4.Lefebvre V, Dumitriu B, Penzo-Mendez A, Han Y, Pallavi B. Control of cell fate and differentiation by Sry-related high-mobility-group box (Sox) transcription factors. Int J Biochem Cell Biol. 2007;39(12):2195–214. Epub 2007/07/13. doi: 10.1016/j.biocel.2007.05.019 ; PubMed Central PMCID: PMC2080623. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0298818.ref005] 5.Kamachi Y, Cheah KS, Kondoh H. Mechanism of regulatory target selection by the SOX high-mobility-group domain proteins as revealed by comparison of SOX1/2/3 and SOX9. Mol Cell Biol. 1999;19(1):107–20. Epub 1998/12/22. doi: 10.1128/MCB.19.1.107 ; PubMed Central PMCID: PMC83870. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0298818.ref006] 6.Phochanukul N, Russell S. No backbone but lots of Sox: Invertebrate Sox genes. Int J Biochem Cell Biol. 2010;42(3):453–64. Epub 2009/07/11. doi: 10.1016/j.biocel.2009.06.013 . [DOI] [PubMed] [Google Scholar]

[pone.0298818.ref007] 7.Soriano NS, Russell S. The Drosophila SOX-domain protein Dichaete is required for the development of the central nervous system midline. Development. 1998;125(20):3989–96. Epub 1998/09/15. doi: 10.1242/dev.125.20.3989 . [DOI] [PubMed] [Google Scholar]

[pone.0298818.ref008] 8.Uchikawa M, Kamachi Y, Kondoh H. Two distinct subgroups of Group B Sox genes for transcriptional activators and repressors: their expression during embryonic organogenesis of the chicken. Mech Dev. 1999;84(1–2):103–20. Epub 1999/09/03. doi: 10.1016/s0925-4773(99)00083-0 . [DOI] [PubMed] [Google Scholar]

[pone.0298818.ref009] 9.Pevny L, Placzek M. SOX genes and neural progenitor identity. Current opinion in neurobiology. 2005;15(1):7–13. Epub 2005/02/22. doi: 10.1016/j.conb.2005.01.016 . [DOI] [PubMed] [Google Scholar]

[pone.0298818.ref010] 10.Cremazy F, Berta P, Girard F. Sox neuro, a new Drosophila Sox gene expressed in the developing central nervous system. Mechanisms of development. 2000;93(1–2):215–9. doi: 10.1016/s0925-4773(00)00268-9 . [DOI] [PubMed] [Google Scholar]

[pone.0298818.ref011] 11.Hardcastle Z, Papalopulu N. Distinct effects of XBF-1 in regulating the cell cycle inhibitor p27(XIC1) and imparting a neural fate. Development. 2000;127(6):1303–14. doi: 10.1242/dev.127.6.1303 . [DOI] [PubMed] [Google Scholar]

[pone.0298818.ref012] 12.Holland LZ, Schubert M, Holland ND, Neuman T. Evolutionary conservation of the presumptive neural plate markers AmphiSox1/2/3 and AmphiNeurogenin in the invertebrate chordate amphioxus. Developmental biology. 2000;226(1):18–33. doi: 10.1006/dbio.2000.9810 . [DOI] [PubMed] [Google Scholar]

[pone.0298818.ref013] 13.Mizuseki Kishi, Matsui Nakanishi, Sasai. Xenopus Zic-related-1 and Sox-2, two factors induced by chordin, have distinct activities in the initiation of neural induction. Development. 1998;125(4):579–87. doi: 10.1242/dev.125.4.579 [DOI] [PubMed] [Google Scholar]

[pone.0298818.ref014] 14.Penzel R, Oschwald R, Chen Y, Tacke L, Grunz H. Characterization and early embryonic expression of a neural specific transcription factor xSOX3 in Xenopus laevis. Int J Dev Biol. 1997;41(5):667–77. . [PubMed] [Google Scholar]

[pone.0298818.ref015] 15.Rex M, Orme A, Uwanogho D, Tointon K, Wigmore PM, Sharpe PT, et al. Dynamic expression of chicken Sox2 and Sox3 genes in ectoderm induced to form neural tissue. Dev Dyn. 1997;209(3):323–32. Epub 1997/07/01. doi: . [DOI] [PubMed] [Google Scholar]

[pone.0298818.ref016] 16.Russell SR, Sanchez-Soriano N, Wright CR, Ashburner M. The Dichaete gene of Drosophila melanogaster encodes a SOX-domain protein required for embryonic segmentation. Development. 1996;122(11):3669–76. doi: 10.1242/dev.122.11.3669 . [DOI] [PubMed] [Google Scholar]

[pone.0298818.ref017] 17.Streit A, Sockanathan S, Perez L, Rex M, Scotting PJ, Sharpe PT, et al. Preventing the loss of competence for neural induction: HGF/SF, L5 and Sox-2. Development. 1997;124(6):1191–202. doi: 10.1242/dev.124.6.1191 . [DOI] [PubMed] [Google Scholar]

[pone.0298818.ref018] 18.Uwanogho D, Rex M, Cartwright EJ, Pearl G, Healy C, Scotting PJ, et al. Embryonic expression of the chicken Sox2, Sox3 and Sox11 genes suggests an interactive role in neuronal development. Mech Dev. 1995;49(1–2):23–36. Epub 1995/01/01. doi: 10.1016/0925-4773(94)00299-3 . [DOI] [PubMed] [Google Scholar]

[pone.0298818.ref019] 19.Pevny LH, Sockanathan S, Placzek M, Lovell-Badge R. A role for SOX1 in neural determination. Development. 1998;125(10):1967–78. Epub 1998/06/18. doi: 10.1242/dev.125.10.1967 . [DOI] [PubMed] [Google Scholar]

[pone.0298818.ref020] 20.Bylund M, Andersson E, Novitch BG, Muhr J. Vertebrate neurogenesis is counteracted by Sox1-3 activity. Nat Neurosci. 2003;6(11):1162–8. Epub 2003/10/01. doi: 10.1038/nn1131 . [DOI] [PubMed] [Google Scholar]

[pone.0298818.ref021] 21.Graham V, Khudyakov J, Ellis P, Pevny L. SOX2 functions to maintain neural progenitor identity. Neuron. 2003;39(5):749–65. Epub 2003/09/02. doi: 10.1016/s0896-6273(03)00497-5 . [DOI] [PubMed] [Google Scholar]

[pone.0298818.ref022] 22.Kondo T, Raff M. Chromatin remodeling and histone modification in the conversion of oligodendrocyte precursors to neural stem cells. Genes Dev. 2004;18(23):2963–72. doi: 10.1101/gad.309404 ; PubMed Central PMCID: PMC534656. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0298818.ref023] 23.Wegner M, Stolt CC. From stem cells to neurons and glia: a Soxist’s view of neural development. Trends Neurosci. 2005;28(11):583–8. doi: 10.1016/j.tins.2005.08.008 . [DOI] [PubMed] [Google Scholar]

[pone.0298818.ref024] 24.Cavallaro M, Mariani J, Lancini C, Latorre E, Caccia R, Gullo F, et al. Impaired generation of mature neurons by neural stem cells from hypomorphic Sox2 mutants. Development. 2008;135(3):541–57. doi: 10.1242/dev.010801 . [DOI] [PubMed] [Google Scholar]

[pone.0298818.ref025] 25.Fantes J, Ragge NK, Lynch SA, McGill NI, Collin JR, Howard-Peebles PN, et al. Mutations in SOX2 cause anophthalmia. Nat Genet. 2003;33(4):461–3. Epub 2003/03/04. doi: 10.1038/ng1120 . [DOI] [PubMed] [Google Scholar]

[pone.0298818.ref026] 26.Sisodiya SM, Ragge NK, Cavalleri GL, Hever A, Lorenz B, Schneider A, et al. Role of SOX2 mutations in human hippocampal malformations and epilepsy. Epilepsia. 2006;47(3):534–42. doi: 10.1111/j.1528-1167.2006.00464.x . [DOI] [PubMed] [Google Scholar]

[pone.0298818.ref027] 27.Hagey DW, Muhr J. Sox2 acts in a dose-dependent fashion to regulate proliferation of cortical progenitors. Cell reports. 2014;9(5):1908–20. doi: 10.1016/j.celrep.2014.11.013 . [DOI] [PubMed] [Google Scholar]

[pone.0298818.ref028] 28.Chew LJ, Gallo V. The Yin and Yang of Sox proteins: Activation and repression in development and disease. J Neurosci Res. 2009;87(15):3277–87. doi: 10.1002/jnr.22128 ; PubMed Central PMCID: PMC2767240. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0298818.ref029] 29.Liu YR, Laghari ZA, Novoa CA, Hughes J, Webster JR, Goodwin PE, et al. Sox2 acts as a transcriptional repressor in neural stem cells. BMC Neurosci. 2014;15:95. doi: 10.1186/1471-2202-15-95 ; PubMed Central PMCID: PMC4148960. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0298818.ref030] 30.Remenyi A, Lins K, Nissen LJ, Reinbold R, Scholer HR, Wilmanns M. Crystal structure of a POU/HMG/DNA ternary complex suggests differential assembly of Oct4 and Sox2 on two enhancers. Genes Dev. 2003;17(16):2048–59. doi: 10.1101/gad.269303 ; PubMed Central PMCID: PMC196258. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0298818.ref031] 31.Fang L, Zhang L, Wei W, Jin X, Wang P, Tong Y, et al. A methylation-phosphorylation switch determines Sox2 stability and function in ESC maintenance or differentiation. Mol Cell. 2014;55(4):537–51. doi: 10.1016/j.molcel.2014.06.018 . [DOI] [PubMed] [Google Scholar]

[pone.0298818.ref032] 32.Jeong CH, Cho YY, Kim MO, Kim SH, Cho EJ, Lee SY, et al. Phosphorylation of Sox2 cooperates in reprogramming to pluripotent stem cells. Stem Cells. 2010;28(12):2141–50. doi: 10.1002/stem.540 . [DOI] [PubMed] [Google Scholar]

[pone.0298818.ref033] 33.Swaney DL, Wenger CD, Thomson JA, Coon JJ. Human embryonic stem cell phosphoproteome revealed by electron transfer dissociation tandem mass spectrometry. Proc Natl Acad Sci U S A. 2009;106(4):995–1000. doi: 10.1073/pnas.0811964106 ; PubMed Central PMCID: PMC2633571. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0298818.ref034] 34.Van Hoof D, Munoz J, Braam SR, Pinkse MW, Linding R, Heck AJ, et al. Phosphorylation dynamics during early differentiation of human embryonic stem cells. Cell Stem Cell. 2009;5(2):214–26. doi: 10.1016/j.stem.2009.05.021 . [DOI] [PubMed] [Google Scholar]

[pone.0298818.ref035] 35.Zhao HY, Zhang YJ, Dai H, Zhang Y, Shen YF. CARM1 mediates modulation of Sox2. PLoS One. 2011;6(10):e27026. Epub 2011/10/28. doi: 10.1371/journal.pone.0027026 ; PubMed Central PMCID: PMC3203945. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0298818.ref036] 36.Baltus GA, Kowalski MP, Zhai H, Tutter AV, Quinn D, Wall D, et al. Acetylation of sox2 induces its nuclear export in embryonic stem cells. Stem Cells. 2009;27(9):2175–84. doi: 10.1002/stem.168 . [DOI] [PubMed] [Google Scholar]

[pone.0298818.ref037] 37.Tsuruzoe S, Ishihara K, Uchimura Y, Watanabe S, Sekita Y, Aoto T, et al. Inhibition of DNA binding of Sox2 by the SUMO conjugation. Biochem Biophys Res Commun. 2006;351(4):920–6. Epub 2006/11/14. doi: 10.1016/j.bbrc.2006.10.130 . [DOI] [PubMed] [Google Scholar]

[pone.0298818.ref038] 38.Rosonina E, Akhter A, Dou Y, Babu J, Sri Theivakadadcham VS. Regulation of transcription factors by sumoylation. Transcription. 2017;8(4):220–31. Epub 2017/04/06. doi: 10.1080/21541264.2017.1311829 ; PubMed Central PMCID: PMC5574528. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0298818.ref039] 39.Kondoh H, Lovell-Badge R. Sox2 Biology and Role in Development and Disease: Academic Press; 2016. [Google Scholar]

[pone.0298818.ref040] 40.Wei F, Scholer HR, Atchison ML. Sumoylation of Oct4 enhances its stability, DNA binding, and transactivation. The Journal of biological chemistry. 2007;282(29):21551–60. Epub 2007/05/26. doi: 10.1074/jbc.M611041200 . [DOI] [PubMed] [Google Scholar]

[pone.0298818.ref041] 41.Zhang Z, Liao B, Xu M, Jin Y. Post-translational modification of POU domain transcription factor Oct-4 by SUMO-1. FASEB J. 2007;21(12):3042–51. Epub 2007/05/15. doi: 10.1096/fj.06-6914com . [DOI] [PubMed] [Google Scholar]

[pone.0298818.ref042] 42.Taylor KM, Labonne C. SoxE factors function equivalently during neural crest and inner ear development and their activity is regulated by SUMOylation. Dev Cell. 2005;9(5):593–603. Epub 2005/11/01. doi: 10.1016/j.devcel.2005.09.016 . [DOI] [PubMed] [Google Scholar]

[pone.0298818.ref043] 43.Wegner M. From head to toes: the multiple facets of Sox proteins. Nucleic Acids Res. 1999;27(6):1409–20. Epub 1999/02/26. doi: 10.1093/nar/27.6.1409 ; PubMed Central PMCID: PMC148332. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0298818.ref044] 44.Bowles J, Schepers G, Koopman P. Phylogeny of the SOX family of developmental transcription factors based on sequence and structural indicators. Dev Biol. 2000;227(2):239–55. Epub 2000/11/10. doi: 10.1006/dbio.2000.9883 . [DOI] [PubMed] [Google Scholar]

[pone.0298818.ref045] 45.Savare J, Bonneaud N, Girard F. SUMO represses transcriptional activity of the Drosophila SoxNeuro and human Sox3 central nervous system-specific transcription factors. Mol Biol Cell. 2005;16(6):2660–9. Epub 2005/03/25. doi: 10.1091/mbc.e04-12-1062 ; PubMed Central PMCID: PMC1142414. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0298818.ref046] 46.Kuo CL, Lam CM, Hewitt JE, Scotting PJ. Formation of the embryonic organizer is restricted by the competitive influences of Fgf signaling and the SoxB1 transcription factors. PLoS One. 2013;8(2):e57698. Epub 2013/02/28. doi: 10.1371/journal.pone.0057698 ; PubMed Central PMCID: PMC3585176. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0298818.ref047] 47.Shih YH, Kuo CL, Hirst CS, Dee CT, Liu YR, Laghari ZA, et al. SoxB1 transcription factors restrict organizer gene expression by repressing multiple events downstream of Wnt signalling. Development. 2010;137(16):2671–81. doi: 10.1242/dev.054130 . [DOI] [PubMed] [Google Scholar]

[pone.0298818.ref048] 48.Zhang C, Basta T, Jensen ED, Klymkowsky MW. The beta-catenin/VegT-regulated early zygotic gene Xnr5 is a direct target of SOX3 regulation. Development. 2003;130(23):5609–24. Epub 2003/10/03. doi: 10.1242/dev.00798 . [DOI] [PubMed] [Google Scholar]

[pone.0298818.ref049] 49.Li J, Pan G, Cui K, Liu Y, Xu S, Pei D. A dominant-negative form of mouse SOX2 induces trophectoderm differentiation and progressive polyploidy in mouse embryonic stem cells. J Biol Chem. 2007;282(27):19481–92. doi: 10.1074/jbc.M702056200 . [DOI] [PubMed] [Google Scholar]

[pone.0298818.ref050] 50.Seo E, Basu-Roy U, Zavadil J, Basilico C, Mansukhani A. Distinct functions of Sox2 control self-renewal and differentiation in the osteoblast lineage. Mol Cell Biol. 2011;31(22):4593–608. Epub 2011/09/19. doi: 10.1128/MCB.05798-11 ; PubMed Central PMCID: PMC3209254. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0298818.ref051] 51.Vickers ER, Sharrocks AD. The use of inducible engrailed fusion proteins to study the cellular functions of eukaryotic transcription factors. Methods. 2002;26(3):270–80. doi: 10.1016/S1046-2023(02)00031-2 . [DOI] [PubMed] [Google Scholar]

[pone.0298818.ref052] 52.Ross S, Best JL, Zon LI, Gill G. SUMO-1 modification represses Sp3 transcriptional activation and modulates its subnuclear localization. Mol Cell. 2002;10(4):831–42. doi: 10.1016/s1097-2765(02)00682-2 . [DOI] [PubMed] [Google Scholar]

[pone.0298818.ref053] 53.Holmstrom S, Van Antwerp ME, Iñiguez-Lluhi JA. Direct and distinguishable inhibitory roles for SUMO isoforms in the control of transcriptional synergy. Proc Natl Acad Sci U S A. 2003;100(26):15758–63. Epub 2003/12/08. doi: 10.1073/pnas.2136933100 ; PubMed Central PMCID: PMC307641. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0298818.ref054] 54.Long J, Wang G, He D, Liu F. Repression of Smad4 transcriptional activity by SUMO modification. Biochem J. 2004;379(Pt 1):23–9. doi: 10.1042/BJ20031867 ; PubMed Central PMCID: PMC1224064. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0298818.ref055] 55.Wu F, Zhang J, Wang P, Ye X, Jung K, Bone KM, et al. Identification of two novel phenotypically distinct breast cancer cell subsets based on Sox2 transcription activity. Cell Signal. 2012;24(11):1989–98. doi: 10.1016/j.cellsig.2012.07.008 . [DOI] [PubMed] [Google Scholar]

[pone.0298818.ref056] 56.Wu Y, Guo Z, Wu H, Wang X, Yang L, Shi X, et al. SUMOylation represses Nanog expression via modulating transcription factors Oct4 and Sox2. PLoS One. 2012;7(6):e39606. Epub 2012/06/30. doi: 10.1371/journal.pone.0039606 ; PubMed Central PMCID: PMC3382131. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0298818.ref057] 57.Chymkowitch P, Nguea PA, Enserink JM. SUMO-regulated transcription: challenging the dogma. Bioessays. 2015;37(10):1095–105. doi: 10.1002/bies.201500065 . [DOI] [PubMed] [Google Scholar]

[pone.0298818.ref058] 58.Hay RT. SUMO: a history of modification. Mol Cell. 2005;18(1):1–12. doi: 10.1016/j.molcel.2005.03.012 . [DOI] [PubMed] [Google Scholar]

[pone.0298818.ref059] 59.Yau TY, Molina O, Courey AJ. SUMOylation in development and neurodegeneration. Development. 2020;147(6). Epub 20200318. doi: 10.1242/dev.175703 ; PubMed Central PMCID: PMC7097199. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0298818.ref060] 60.Thiel G. How Sox2 maintains neural stem cell identity. The Biochemical journal. 2013;450(3):e1–2. Epub 2013/03/01. doi: 10.1042/BJ20130176 . [DOI] [PubMed] [Google Scholar]

[pone.0298818.ref061] 61.Liebelt F, Vertegaal AC. Ubiquitin-dependent and independent roles of SUMO in proteostasis. Am J Physiol Cell Physiol. 2016;311(2):C284–96. Epub 2016/06/24. doi: 10.1152/ajpcell.00091.2016 ; PubMed Central PMCID: PMC5129774. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0298818.ref062] 62.Qi Y, Liu H, Lin X. Sumoylation Stabilizes Smoothened to Promote Hedgehog Signaling. Dev Cell. 2016;39(4):385–7. Epub 2016/11/23. doi: 10.1016/j.devcel.2016.11.001 . [DOI] [PubMed] [Google Scholar]

[pone.0298818.ref063] 63.Wen D, Wu J, Wang L, Fu Z. SUMOylation Promotes Nuclear Import and Stabilization of Polo-like Kinase 1 to Support Its Mitotic Function. Cell reports. 2017;21(8):2147–59. Epub 2017/11/23. doi: 10.1016/j.celrep.2017.10.085 ; PubMed Central PMCID: PMC5728694. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0298818.ref064] 64.Homem CC, Repic M, Knoblich JA. Proliferation control in neural stem and progenitor cells. Nat Rev Neurosci. 2015;16(11):647–59. Epub 2015/10/01. doi: 10.1038/nrn4021 ; PubMed Central PMCID: PMC4667397. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0298818.ref065] 65.Cossec JC, Theurillat I, Chica C, Bua Aguin S, Gaume X, Andrieux A, et al. SUMO Safeguards Somatic and Pluripotent Cell Identities by Enforcing Distinct Chromatin States. Cell stem cell. 2018;23(5):742–57 e8. Epub 2018/11/08. doi: 10.1016/j.stem.2018.10.001 . [DOI] [PubMed] [Google Scholar]

PERMALINK

SUMO-dependent transcriptional repression by Sox2 inhibits the proliferation of neural stem cells

Elisa Marelli

Jaime Hughes

Paul J Scotting

Roles

Abstract

Introduction

Materials and methods

Design and cloning of Sox2 mutant constructs

Western blot

Subcellular fractionation

Culture and transfection of hNSC

Luciferase reporter assay

RNA sequencing and data analysis

Cell proliferation assay

Cell death assay

Results

Sox2 SUMOylation inhibits its transcriptional activation ability

Fig 1. Luciferase reporter assay performed on HEK-293 over-expressing different Sox2 mutant constructs.

Fig 2. Schematic representation of Sox2 SUMOylation mutant constructs.

SUMOylation of Sox2 is necessary for the protein’s transcriptional repression activity

Fig 3. Comparison of differentially expressed genes in hNSC over-expressing wt Sox2, Sox2_K247R or Sox2_K247R-SUMO2.

SUMO-dependent Sox2 repressed target genes are involved in the regulation of the cell cycle

Table 1. Results of gene ontology analysis of genes down-regulated in hNSC over-expressing wt Sox2 or Sox2_K247R-SUMO2 based on cellular process.

Table 2. Results of gene ontology analysis of genes down-regulated in hNSC over-expressing wt Sox2 or Sox2_K247R-SUMO2 based on molecular function.

Fig 4. Gene ontology analysis performed on genes differentially expressed when wt Sox2 and Sox2_K247R are over-expressed.

SUMOylation of Sox2 affects hNSC proliferation

Fig 5. Proliferation over the course of 7 days of hNSC transiently transfected with either pcDNA3 (empty vector), wt Sox2, Sox2_K247R or Sox2_K247R-SUMO2.

Fig 6. Comparison between proliferation rates of hNSC transfected with empty plasmid (pcDNA3), wt Sox2, Sox2_K247R or Sox2_K247R-SUMO2 and cultured for 7 days.

SUMOylation does not affect apoptosis in hNSC

Fig 7. Cell death of hNSC treated with G418 or transfected with either empty vector (pcDNA3) or different Sox2 constructs.

SUMOylation does not affect Sox2 subcellular localisation

Fig 8. Quantitative analysis of wt Sox2, Sox2_K247R and Sox2_K247R-SUMO2 across sub-cellular compartments as measured from infrared fluorescence western blot imaging.

Discussion

Supporting information

Data Availability

Funding Statement

References

Decision Letter 0

Anujith Kumar

Roles

Author response to Decision Letter 0

Decision Letter 1

Anujith Kumar

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Anujith Kumar

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